Microsporogenesis, Megasporogenesis, and the Development of Male and Female Gametophytes in Eustoma Grandiflorum

Microsporogenesis, Megasporogenesis, and the Development of Male and Female Gametophytes in Eustoma grandiflorum

Xinyu Yang1, Qiuhong Wang1,2 and Yuhua Li1*

1College of Life Sciences, Northeast Forestry University, Harbin 150040, China 2College of Life Sciences, Heilongjiang University, Harbin 150080, China

Embryological characters during microsporogenesis, megasporogenesis, and the development of male and female gametophytes in Eustoma grandiflorum were observed by microscopy. The results are as follows. 1. The formation of anther walls was of the dicotyledonous type. The tapetum was of the heteromorphic and glandular type. Tapetal cells on the connective side elongated radially. 2. Cytokinesis in microsporocyte meiosis was of the simultaneous type and microspore tetrads were tetrahedral. 3. Mature pollen grains were 2-celled and had 3 germ furrows. 4. The ovary was bicarpellary syncarpous and unilocular, having parietal placentas. Ovules were numerous and anatropous. 5. The archespore under the nucellar epidermis directly developed a megaspore mother cell, which in turn underwent meiotic division to form 4 megaspores arranged in a line or T-shape. The chalazal megaspore was observed to be functional. 6. The formation of the embryo sac was of the polygonum type. Before fertilization, the 2 polar nuclei fused into a secondary nucleus. The mature embryo sac was made up of 7 cells. 7. It was found at a very low rate that there were 2 megasporocytes or 2 embryo sacs in an ovule.

Key Words: Eustoma grandiflorum, female gametophyte, male gametophyte, megasporogenesis, microsporogenesis.

several commercial cultivars, such as ‘Danlunzhusha’ Introduction (Registration Number of Heilongjiang Province in China Eustoma grandiflorum (Raf.) Shinn. (lisianthus, is Heidengji2006036) and ‘Qianduixue’ (Registration Russell prairie gentian) is a genus of the Gentianaceae Number is Heidengji2006037). At present, the breeding family (Ohkawa, 1995). In E. grandiflorum, there are of cultivars adaptable to cold climate is under many cultivars producing flowers in diverse colors with consideration in order to more extensively produce various vase lives. It originated in the West Indies, E. grandiflorum in northern China. For the introduction Mexico, and Central and South America. Wild plants of cold adaptable traits, intergeneric crossing is have blue-purple flowers, but intensive breeding in Japan unavoidable since cold adaptable lines are not available. over the past 30 years has produced cultivars with white, The expected occurrence of cross-incompatibility will pink, and mauve flowers. E. grandiflorum has been used be attempted to be overcome by irradiation with a laser, as a breeding material for a number of cultivars of prairie which was shown to be effective in Brassica (Li and gentian (Okamoto et al., 2002). Much research in Yu, 2006). However, unsuccessful crossings due to E. grandiflorum has been made on heredity and selection events at several developmental stages such as the failure concerning floral color and shape (Jamal Uddin et al., in pollen germination and pollen tube growth and the 2002, 2004). The genetic transformation of related genes abortion of ovules and embryos will be unavoidable. was also conducted (Ledger et al., 1997; Semeria et al., Information on the development of reproductive organs 1996). in E. grandiflorum is essential for overcoming these. We In our laboratory, we conducted breeding experiments also intend to develop an efficient breeding method of concerning flower traits of E. grandiflorum using this plant by applying the floral-dip method (Bent, 2000; conventional cross-breeding methods and registed Clough et al., 1998; Desfeux et al., 2000), in which a more exact judgment of the developmental stage of ovules in flower buds is crucial. Received; February 13, 2006. Accepted; November 8, 2006. This work was supported by the Natural Science Foundation of China The study of microsporogenesis, megasporogenesis, (NSFC) (project 30371189). and the development of male and female gametophytes * Corresponding author (E-mail: [email protected]). in E. grandiflorum will provide basic information for

244 J. Japan. Soc. Hort. Sci. 76 (3): 244–249. 2007. 245 our research projects. Landmann (1958) (Fig. 1g). Tapetal cells were uninucleate with 2 nucleoli, each of which showed a round Materials and Methods or ellipsoidal nucleus with a dense cytoplasm throughout The breeding line ‘02-49’ of E. grandiflorum with development (Fig. 1e–g). At about the time of meiosis single yellow flowers was used. Seeds were sown in of a microspore mother cell, tapetal cells showed February 2005 in a greenhouse at the Research Institute indistinct walls and appeared to be degenerating of Flower Biotechnology, Northeast Forestry University, (Fig. 1h). At the stage of one-nucleate free microspores China. In September, more than 100 flower buds or and two-nucleate pollen grains, tapetal cells degenerated flowers on main shoots or the first lateral branches were completely. The tapetal cells degenerated at their original taken at each of seven growth stages evaluated by the sites. Therefore, the tapetum was of the heteromorphic, length of buds, i.e., less than 0.5 cm, 0.5 to less than named firstly by Bhojwani and Bhatnagar (1979), and 1 cm, 1 to less than 1.5 cm, 1.5 to less than 2 cm, 2 to glandular type. less than 2.5 cm, 2.5 to less than 3 cm, and 3 to less than 3.5 cm. The size of buds was measured in terms of the Microsporogenesis and microgametogenesis length between the top and base with the naked eye or Simultaneously with changes taking place in the wall a microscope, and that of ovaries between the stylar base of the microsporangia, the primary sporogenous cells and the bottom of an ovary. The anthers and ovaries underwent mitosis to form secondary sporogenous cells, were fixed in FAA (5 mL formalin: 6 mL acetic acid: from which microsporocytes were derived (Fig. 1i). 89 mL 70% (v/v) ethanol). After being stained with Microsporocytes were recognizable by their large Ehrfich’s haematoxylin, tissues were embedded in volume, dense cytoplasm, and conspicuous nuclei. The paraffin using a conventional method, and cut at a microsporocyte underwent meiosis, and the process of thickness of 6 to 10 µm. Observation and photomicros- meiosis involved 2 cell divisions. Meiosis I includes the copy of sections were carried out using a microscope prophase, which is sub-staged as leptotene (Fig. 1j), (BX15, Olympus, Japan). amphitene, pachytene, diplotene, and diakinasis (Fig. 1k), metaphase (Fig. 1l), anaphase, and telophase Results (Fig. 1m). As a result, a microspore dyad is formed. A Formation of anther wall microspore tetrad was formed via the prophase, Flowers of E. grandiflorum were bisexual and metaphase (Fig. 1n, o), anaphase, and telophase. There protandrous. Mature flowers had a calyx of 5 sepals, a were 2 kinds of microspore tetrads: tetrahedral tetrads corolla of 5 petals, 5 stamens attached on the corolla occupying 95% of observed microspore tetrads throat, and a single-celled ovary with 2 stigmata, similar (Fig. 1p, r) and decussate tetrads at 5% (Fig. 1q). There to the results of Bailey (1950). The anthers were was a thick callous wall outside a microspore tetrad tetrasporangiate. At an early stage of development, (Fig. 1p). As the microspore developed, callous walls archesporial cells, which were recognizable by their large disappeared (Fig. 1q, r). During meiosis, cytokinesis was volume and conspicuous nuclei, differentiated below the of the simultaneous type (Fig. 1m–r). epidermis of anthers (Fig. 1a). These cells divided Free uninucleate microspores were separated from the periclinally to form outer primary parietal cells and inner tetrad. Each microspore had a dense cytoplasm, primary sporogenous cells (Fig. 1b). The primary parietal conspicuous wall, and a prominent and centrally placed cells divided periclinally and anticlinally to form 2 layers nucleus (Fig. 1s). As the central vacuole developed, the of secondary parietal cells (Fig. 1c). The outer secondary nucleus took a peripheral position (Fig. 1t). At this stage, parietal cells formed a subepidermal endothecium and the microspore was uninucleate and peripheral. Mitosis two middle layers by periclinal and anticlinal divisions (Fig. 1u) of the microspore nucleus resulted in the (Fig. 1d, e). The middle layers had a common formation of 2 unequal cells, i.e., a larger vegetative and histogenetic origin with the endothecium (Fig. 1d, e). a smaller generative one (Fig. 1v). Then, the generative The inner secondary parietal cells gave rise to the cell was submerged in the cytoplasm of a vegetative cell tapetum, which partly originated from the ground tissue (Fig. 1w). Mature pollen grains were two-celled near the connective tissue (Fig. 1f, g). Thus, the tapetum (Fig. 1x). was of dual origin. The microsporangial wall formation was therefore of the dicotyledonous type. The Megasporogenesis and megagametogenesis endothecium comprised 1 layer. The middle layers There were approximately 2000 to 3000 ovules in 4 appeared to undergo further divisions to form 3 layers. rows in the placenta of an ovary in E. grandiflorum. The The anther wall prior to maturation was usually ovary was superior, bicarpellate, and unilocular with comprised of 6 layers, i.e., an epidermis, an endothecium, parietal placentae. The integument was initiated by 3 middle layers, and a layered tapetum (Fig. 1e–g). periclinal and oblique divisions at the base of the Cells of the tapetum on the connective side showed nucellus. The ovule was unitegmental. The integument radial elongation and intruded into the anther locule to reached the top of the nucellus and formed a micropyle form ‘placentoids’, which were named by Steffen and by continued division. Ovules in E. grandiflorum 246 X. Yang, Q. Wang and Y. Li

Fig. 1. Formation of the anther wall, microsporogenesis, and microgametogenesis in Eustoma grandiflorum. a. Archesporial cells. The arrow indicates an archesporial cell. b. Periclinal division of archesporial cells. c. Sporogenous cells and secondary parietal cells beneath the epiderm. d. The outermost secondary parietal cells divided the endothecium (end) and middle layer (ml), showing the dicotyledonous type of wall formation. The arrows refer to the four layers from the left to right: the epiderm (epi), endothecium (end), and two middle layers (ml). e and f. Anther wall composed of the epidermis (epi), endothecium (end), middle layer (ml), and tapetum (tap). The arrows refer to the six layers from the left to right: the epiderm (epi), endothecium (end), three middle layers (ml), and the tapetum (tap). The microspores (mic) are in the middle. g. Heteromorphic and glandular tapetum. The arrows delineate the tapetum (tap) on the two sides. h. Degenerated tapetal cells. The arrow shows a degenerated tapetal cell (tap). i. Microspore mother cells (mmc). The arrow refers to the tepetal cell (tap) and a microspore mother cell (mmc). j. Meiosis of the microsporocyte, leptotene in prophase. k. Diakinasis in prophase. l. Metaphase. m. Telophase. n and o. Metaphase. p and q. Tetrahedral and decussate tetrad. The arrow refers to the thick callous wall outside a microspore tetrad. r. Degenerated callous wall. s. Free uninucleate microspores. t. Uninucleate peripheral stage of microspore. u. Telophase of microspore mitosis. v. Vegetative cells and generative cells. The arrows refer to the vegetative cell and generative cell. w. The generative cell was submerged in the cytoplasm of large vegetative cells. The arrows refer to the generative cell and the nucleus of the vegetative cell. x. Two- celled pollen grains. The arrows refer to the generative cell and the nucleus of the vegetative cell. Scale bar = 15 µm for a to f and i, 80 µm for g and h, 10 µm for j to x. J. Japan. Soc. Hort. Sci. 76 (3): 244–249. 2007. 247

Fig. 2. Megasporogenesis and megagametogenesis in Eustoma grandiflorum. a. Single archesporial cells as a megasporocyte, tenuinucellate. The arrow refers to the archesporial cell. b. Meiosis of the megasporocyte, leptotene in prophase. c. Amphitene in prophase. d. Diakinasis in prophase. e. Metaphase. f. Anaphase. g. Prophase. h and i. Metaphase. j. Linear tetrad of megaspores. The arrows indicate the three degenerated micropylar megaspores (dm) and a functional megaspore (fm) at the chalazal end. k. Three degenerated micropylar megaspores of a tetrad (dm), and the functional megaspore (fm) at the chalazal end. The arrows refer to the three degenerated micropylar megaspores (dm) and a functional megaspore (fm) at the chalazal end. l and m. A bi-nucleate embryo sac. n. Four-nucleate embryo sac. o. Five nuclei of an eight-nucleate embryo sac. The arrows refer to five nuclei of an eight-nucleate embryo sac. p. Two polar nuclei (pn). The arrows refer to the two polar nuclei (pn). q and r. Three micropylar nuclei became the egg (en: egg nucleus) and 2 synergids (sy), and comprised the egg apparatus. The chalazal nuclei became 3 antipodals (ant). The polar nuclei fused at the center and the resulting fusion moved close to the egg apparatus. The arrow indicates the egg nucleus (en). s. 2 archesporial cells. t. 2 embryo sacs. Scale bar = 10 µm for a to k, p, q and s, 20 µm for l to o, r and t. 248 X. Yang, Q. Wang and Y. Li

Table 1. The relationship between the size of flower buds or flowers and the development of pistils or stamens in E. grandiflorum.

Length of flower buds and an ovary Developmental event in pistils Developmental event in stamens 0.2–0.3 cm bud Ovule primordium Archesporium 0.6–0.7 cm bud Ovary primordium Microspore mother cells 0.7–1 cm bud and 0.3 cm ovary Archesporium Microspore tetrads 1–1.6 cm bud and 0.3–0.5 cm ovary Meiosis of megasporocytes Mononuclear microspores 1.8–2.6 cm bud and 0.6–0.8 cm ovary Development of embryo sacs Two-celled pollen 3.5 cm bud and 1.0–1.2 cm ovary at anthesis Mature embryo sacs Pollen grains released were anatropous. length was 0.7–1.0 cm with an ovary of 0.3 cm in length, A single archesporial cell (Fig. 2a) differentiated under the developmental event in pistils was the archesporium one-layered epidermal cells in the young nucellus. This and that in stamens was microspore tetrads. When the archesporial cell functioned directly as the megasporo- bud length was 1.0–1.6 cm with an ovary of 0.3–0.5 cm, cyte, which was characterized by a large nucleus and the stage was at the meiosis of megasporocytes in pistils dense cytoplasm. Thus, the ovule was tenuinucellate and mononuclear microspores in stamens. For a bud of (Fig. 2b–d). It was found at a low rate that there were 2 1.8–2.6 cm in length with an ovary of 0.6–0.8 cm, the archesporial cells in an ovule (Fig. 2s). development of embryo sacs and two-celled pollen were The megasporocyte underwent meiosis to form a observed. During anthesis, the bud length was 3.5 cm megaspore dyad. Meiosis I includes the prophase, which with an ovary of 1.0–1.2 cm, and the ovary was at the is sub-staged as leptotene (Fig. 2b), amphitene (Fig. 2c), stage of mature embryo sacs. pachytene, diplotene, and diakinasis (Fig. 2d), metaphase Discussion (Fig. 2e), anaphase (Fig. 2f), and telophase. Subsequently a megaspore tetrad was formed via the prophase The method of embryogenesis in E. grandiflorum was (Fig. 2g), metaphase (Fig. 2h, i), anaphase, and telophase. the same as those reported for other species of Finally, these processes gave rise to a linear tetrad of Gentianaceae in terms of the glandular tapetum, megaspores (Fig. 2j). While 3 micropylar megaspores of simultaneous occurrence of microsporocyte meiosis, the tetrad eventually degenerated, the chalazal one two-celled pollen grains, the tenuinucellate, unitegmen- became functional (Fig. 2k). The functional megaspore tal, and anatropous ovules, and the polygonum-type developed successively into a two- (Fig. 2l, m), four- embryo sac (Bhojwani and Bhatnagar, 1997; Chen et (Fig. 2n), and eight-nucleate embryo sac (Fig. 2o) by 3 al., 2000a, b; Davis, 1966; Ho and Liu, 1999; Ho et al., meiotic divisions. Thus, the mode of embryo sac 2000; Johri, 1992; Liu and Ho, 1996, 1997; Xue et al., formation was of the polygonum type. Three micropylar 1999, 2002a, b; Xue and Li, 2005; Zhu and Shen, 1989). nuclei became an egg and 2 synergids, comprising the The tapetum during development of the anther wall egg apparatus (Fig. 2q, r). The 2 nuclei became polar was of the heteromorphic and glandular type. This type nuclei (Fig. 2p–r). The chalazal nuclei became 3 of tapetum was reported for some plants in Gentianaceae, antipodals (Fig. 2r). The polar nuclei fused at the center in which tapetal cells on the connective side elongated and the resulting secondary nucleus moved close to the radially and intruded into anther locules to form egg apparatus (Fig. 2q, r). In the mature eight-nucleate ‘placentoids’ (Chen et al., 2000a, b; Liu and Ho, 1996, embryo sac, the egg cell is recognized by a nucleus at 1997; Xue et al., 1999, 2002a, b; Xue and Li, 2005; Zhu the chalazal end and a large vacuole at the micropylar and Shen, 1989), so the tapetal cells showed dimorphism end. The 2 synergids were recognized by their nuclei at (Ho and Liu, 1999). At about the time of pollen tetrads, the micropylar end and a large vacuole at the chalazal the walls of the tapetal cells became indistinct and the end (Fig. 2q, r). The 3 antipodal cells were ephemeral tapetal cells degenerated at their original site. The tapetal and degenerated soon after fertilization. Two embryo cells degenerated completely at the stage of 1-nucleate sacs were found in an ovule at a rate of about 0.2% (10 pollen grains and the nucleus near the wall. Thus, the in 5000 ovules) (Fig. 2t). tapetum is heteromorphic and glandular. In view of its structure and function, this kind of tapetum is considered The development of male and female gametophytes in to be more evolutionally advanced than the normal relation to flower size glandular type (Zhu and Shen, 1989). The relationship between the size of flower buds and The relationships between the bud length and flowers, and the developmental stage of male and female developmental stage shown in Table 1 can be used in gametophytes is shown in Table 1. the research of embryogenesis and relative molecular During the stage of the ovule primordium in pistils embryology in E. grandiflorum. During genetic and archesporium in stamens, the bud length was 0.2– transformation by the floral dip method, the ovules are 0.3 cm. At the stage of microspore mother cells in the target cells, and Agrobacterium tumefaciens enters stamens, the bud length was 0.6–0.7 cm. When the bud the ovary before the ovules seal (Bent, 2000). This means J. Japan. Soc. Hort. Sci. 76 (3): 244–249. 2007. 249 that successful transformation relates closely with the Sinica 20: 960–967. morphological structure of the floral organ. It is Ho, T. N. and J. Q. Liu. 1999. Embryology of Gentiana lawrencei suggested that the most appropriate time for transforma- var. farreri. Acta Bot. Bor.-Occid. Sinica 19: 234–240 (In Chinese with English abstract). tion by the floral dip method should be when the length Jamal Uddin, A. F. M., F. Hashimoto, S. Nishimoto, K. Shimizu of a bud is shorter than 1 cm or that of an ovary is less and Y. Sakata. 2002. Flower growth, coloration and petal than 0.3 cm. The developmental event in the pistil at this pigmentation in four lisianthus cultivars. J. Japan. Soc. Hort. time is the archesporium, while the integument has not Sci. 71: 40–47. yet been formed. Thus, the archesporium with only one- Jamal Uddin, A. F. M., F. Hashimoto, T. Miwa, K. Ohbo and Y. layer of nucellar cells outside maybe as good as naked Sakata. 2004. Seasonal variation in pigmentation and in the ovary, so that Agrobacterium tumefaciens will be anthocyanidin phenetics in commercial Eustoma flowers. Sci. Hort. 100: 103–115. able to enter easily. Johri, B. M., D. K. Ambegaokar and P. S. Srivastava. 1992. During megasporogenesis and megagametogenesis, Comparative embryology of angiosperms. Springer, Berlin. cases in which an ovule had 2 archesporial cells (1 in Ledger, S. E., S. C. Deroles, D. G. Manson, J. M. Bradley and about 900 ovules), 2 megasporocytes (2 in about 1100 N. K. Given. 1997. Transformation of lisianthus (Eustoma ovules), 2 functional megaspores (5 in about 1300 grandiflorum). Plant Cell Rep. 16: 853–858. ovules), or 2 embryo sacs (2 in about 1700 ovules) were Li, Y. and X. Yu. 2006. Pollination with laser-irradiated pollens rarely observed. For an ovule having 2 embryo sacs, breaks cross-incompatibility between zicaitai (Brassica campestris var. purpurea) and ornamental kale (B. oleracea each embryo sac was normal, i.e., it was composed of var. acephala) to produce hybrids. Sci. Hort. 108: 397–402. 7 cells and had an egg cell. Since the occurrence of these Liu, J. Q. and T. N. Ho. 1996. The embryological studies of 2 embryo sacs in 1 ovule is probably caused by various Comastoma pulmonarium (Gentianaceae). Acta Phytotaxon. kinds of factors inside and outside of the cells, its Sinica 34: 577–585 (In Chinese with English abstract). cytological mechanism should be further clarified in the Liu, J. Q. and T. N. Ho. 1997. Embryology of Gentianopsis future. paludosa (Fr.) Ma. Acta Biol. Plateau Sinica 13: 31–41 (In Chinese with English abstract). Acknowledgements Ohkawa, K. 1995. Breeding and planting in Eustoma (In Japanese). Seibundo-Shinkosha, Tokyo. The authors wish to thank Prof. JiaHeng Shen, College Okamoto, J., S. Limkaisang, N. Hidenoh and S. Takamatsu. 2002. of life and environment Science, Harbin Normal Powdery mildew of prairie gentian: Characteristics, molecular University, China, for his technical assisstance. phylogeny and pathogenicity. J. Gen. Plant Pathol. 68: 200– 207. Literature Cited Semeria L., B. Ruffoni, M. Rabaglio, A. Genga, A. M. Vaira, G. Bailey, L. H. 1950. Eustoma. p. 1176. In: Bailey, L. H. (ed.). The P. Accotto and A. Allavena. 1996. Genetic transformation of standard encyclopedia of horticulture. Macmillan, New York. Eustoma grandiflorum by Agrobacterium tumefaciens. Plant Bent, A. F. 2000. Arabidopsis in planta transformation. Uses, Cell Tiss. Organ Cult. 47: 67–72. mechanisms, and prospects for transformation of other Steffen, K. and W. Landmann. 1958. Entwicklungs geschichtliche species. Plant Physiol. 124: 1540–1547. und cytologische Untersuchungen am Balken-tapetum von Bhojwani, S. S. and S. P. Bhatnagar. 1979. The embryology of Gentiana cruciata und Impatiens glandulifera. Planta 50: angiosperms. 3rd ed., Publ. House, New Delhi. 423–460. Chen, S. L., T. N. Ho, J. Q. Liu and D. Y. Hong. 2000a. Xue, C. Y., T. N. Ho and J. Q. Liu. 1999. Embryology of Swertia Embryology of Crawfurdia delavayi (Gentianaceae) and its tetraptera Maxim. (Gentianaceae) and its systematic systematic value. Israel J. Plant Sci. 48: 113–119. implications. Acta Phytotaxon. Sinica 37: 259–263. Chen, S. L., T. N. Ho, J. Q. Liu and D. Y. Hong. 2000b. Xue, C. Y., T. N. Ho and D. Z. Li. 2002a. Embryology of Swertia Embryology of Tripterospermum cordatum (Gentianaceae). cincta (Gentianaceae) and its systematic value. Acta Bot. Acta Bot. Yunnan 22: 53–58. Yunnan 24: 75–81. Clough, S. J. and A. F. Bent. 1998. Floral dip: a simplified method Xue, C. Y., T. N. Ho and D. Z. Li. 2002b. Megasporogenesis and for Agrobacterium-mediated transformation of Arabidopsis female gametogenesis development of the Tibetan medicine thaliana. Plant J. 16: 735–743. ‘Zang Yin Chen’—Swertia mussotti. Guihaia 22: 249–251. Davis, G. L. 1966. Systematic embryology of the angiosperms. Xue, C. Y. and D. Z. Li. 2005. Embryology of Megacodon John Wiley & Sons Inc., New York. stylophorus and Veratrilla Baillonii (Gentianaceae): descrip- Desfeux, C., S. J. Clough and A. F. Bent. 2000. Female tions and systematic implications. Bot. J. Linnean Soc. 147: reproductive tissues are the primary target of Agrobacterium- 317–331. mediated transformation by the Arabidopsis floral-dip Zhu, X. H. and J. H. Shen. 1989. Microsporogenesis and method. Plant Physiol. 123: 895–904. megasporogenesis, and the development of the male and Ho, T. N., S. L. Chen, J. Q. Liu and D. Y. Hong. 2000. Embryology female gametophytes in Gentiana Manshurica K. Nat. Sci. of Gentiana striata (Gentianaceae). Acta Bot. Bor. Occid. J. Harbin Normal Univ. 5: 63–73.