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(American Journal of Botany. 2001;88:1966-1976.)
© 2001 Botanical Society of America, Inc.

Reproductive Biology

Reproductive biology of henequén (Agave fourcroydes) and its wild ancestor Agave Angustifolia (Agavaceae). i. Gametophyte development1

Nickolai M. Piven2, Felipe A. Barredo-Pool, Ileana C. Borges-Argáez, Miguel A. Herrera-Alamillo, Alberto Mayo-Mosqueda, José L. Herrera-Herrera and Manuel L. Robert

Centro de Investigación Científica de Yucatán, Calle 43, No. 130, Col. Chuburná de Hidalgo, CP 97200, Mérida, Yucatán, México

Received for publication November 7, 2000. Accepted for publication April 3, 2001.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
The pathways of micro- and megagametophyte development in Agave fourcroydes (henequén) and A. angustifolia were studied. We used histology and light microscopy to observe anther ontogeny and ovary differentiation in relation to flower bud size. Both species have the same sexual reproductive strategies and gametophyte development that may be divided into three phases: (1) premeiotic, which includes the establishment of the megaspore mother cell and the pollen mother cell; (2) meiotic, the formation of mature microspores and functional megaspores; (3) postmeiotic, which encompasses the development of mature pollen grains and the formation of the embryo sac. A successive type microsporogenesis was found in both species with formation of T-shaped tetrads and binuclear pollen grains. In vitro germination tests revealed very low pollen fertility. The female gametophyte is formed from two micropylar megaspore cells after the first meiotic division (bisporic type). Male and female gametogenesis occur asynchronously with microsporogenesis finishing before macrosporogenesis. The results so far show that the formation of male and female gametophytes in henequén is affected at different stages and that these alterations might be responsible for the low fertility shown by this species.

Key Words: Agavaceae • anther • embryo sac • henequén • megasporogenesis • microsporogenesis • ovule • pollen


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Agaves are monocotyledonous plants, many of which have their center of origin in Mexico, where some species have been domesticated and are of economic importance as sources of fiber, steroids, spirits, and other products (Gentry, 1982 ; Dahlgren, Clifford, and Yeo, 1985 ; Robert et al., 1992 ; Colunga-García Marín and May-Pat, 1993 ). The only cultivated species in the Yucatan Peninsula is henequén (Agave fourcroydes Lem.) that descends from the wild ancestor Agave angustifolia Haw. (Colunga-García Marín and May-Pat, 1997 ; Colunga-García Marín et al., 1999 ). Both of these agaves are monocarpic perennials that produce flowers only once toward the end of their life cycle of ~20 yr, after which they die. Throughout its life span, henequén propagates mainly by means of its rhizomes whose apical meristems emerge at a distance from the parent plant, giving rise to new individuals. The flowers develop at the top of large inflorescences that can reach 3–8 m high and, after flowering, bulbils originate from buds beneath bracteoles on the inflorescence. However, in spite of their potential utility, bulbils and seeds are not usually used for commercial propagation and have not been used for breeding processes (Eastmond, Herrera, and Robert, 2000) . The traditional agricultural practice of cutting the henequén inflorescences soon after they begin to develop, in order to preserve the plant a little longer, has also limited the supply of seeds.

Fruits develop abnormally because carpels remain empty, probably due to insufficient pollination, and the few seeds that mature show low germination less than 10%. Both phenomena could be due to the pentaploid chromosome level of the species (5x = 150; Castorena-Sánchez, Escobedo, and Quiroz, 1991), which may be responsible for the low fertility of the species. Conversely, in the wild species A. angustifolia, which is a fertile hexaploid (6x = 180; Castorena-Sánchez, Escobedo, and Quiroz, 1991), seed germination is as high as 73% (Colunga-García Marín et al., 1999 ). This led us to include it in this study for comparative purposes. In spite of its economic importance, conventional breeding techniques have never been applied to henequén, probably because its long life span and low fertility have made it almost impossible to carry out genetic improvement. No attempts have been made to cross henequén with other agave species and no natural hybrids have been reported. The only successful program to hybridize hard fiber-producing agaves was started by Doughty in Tanzania in 1931 (Doughty, 1936 ) and culminated in the release of hybrid 11648 (A. angustifolia x A. amaniensis) x A. amaniensis (Lock, 1962 ).

In order to develop alternative methods for the improvement of henequén, Robert et al. (1987, 1992) used in vitro culture to micropropagate high-yielding individuals and to form a germplasm collection. The micropropagated plants that originated in vitro by shoot multiplication are genetically stable and are performing satisfactorily in the field. However, the genetic homogeneity in the vegetatively propagated plantations is still a problem, and the generation of new genetic variability would be highly desirable in order to prevent susceptibility to microbial diseases and other threats to the plantations. Sexual reproduction offers the best alternative to generate the variability needed for improvement programs. Nevertheless, until reliable knowledge of the sexual reproductive process of agaves in general and of henequén in particular is available, this will remain a difficult task.

This is the first report of morphological and histological studies of the sexual reproductive system of A. fourcroydes and its wild relative A. angustifolia. We analyzed male gametophyte (pollen or microgametophyte) and female gametophyte (embryo sac or megagametophyte) development in both species to understand how gametes are formed and what causes the differences in fertility.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
Plant material
Flowering plants of henequén A. fourcroydes Lemaire (5x = 150) and of its wild ancestor A. angustifolia Haworth (6x = 180) are scarce and the inflorescences had to be collected from a variety of locations (Table 1). Inflorescences of A. angustifolia (variant Bab-ki) were found in deciduous forests around Mérida and in coastal dunes near the port of Sisal. Henequén (Sak-ki) was collected from a private garden in Sisal Puerto, from commercial plantations in Telchac Pueblo, Muna, and Baca, and from the agave collection in the Botanical Garden of the Centro de Investigación Científica de Yucatán, Mérida.


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  		Table 1. Agave species from the Yucatan Peninsula used in this study

 
The environmental conditions in the Yucatan Peninsula during the period of study are presented in Table 1. Day temperatures fluctuated between 30° and 40°C and night temperatures between 22° and 25°C. The photoperiod varied from 11 h of light in February to 14 h in July, while the relative light intensity was ~920–1000 J/m2 during the year.

Histological studies
The collected flower buds were measured and cut into two parts: the upper part, with the anthers, was used to study microsporogenesis and the lower part, containing the ovaries, was used for the analysis of megasporogenesis. Samples were fixed for 48 h in a solution of FAA (Berlin and Miksche, 1976 ) consisting of 40% formaldehyde, glacial acetic acid, 95% ethanol and distilled water, 10 : 5 : 50 : 35 parts, respectively, and then dehydrated through a graded ethanol series before being infiltrated and embedded in plastic resin (JB-4 Glycol Methacrylate, Polysciences, Los Angeles, California, USA). Transverse and longitudinal sections (7–10 µm) were cut with a microtome, stained with toluidine blue, and mounted in Permount (SPI5–500, Fisher Scientific, Fair Lawn, New Jersey, USA). Series of sections of the flower buds, anthers, and ovaries were studied and photographed using a Zeiss compound microscope. A total of 100 anthers and ovaries from different plants were studied.

Estimation of pollen fertility
Pollen fertility was evaluated in two different ways: by acetocarmine staining and through in vitro germination. Apparently "good" pollen is spherical and deeply stained. In order to determine the viability of the pollen grains, in vitro germination by the hanging-drop method was tested on two media: (1) 2% sucrose, 0.05% boric acid, and 0.1–0.2% agar; (2) 2% glucose, 200 mg/L calcium chloride, 0.06% boric acid, 2 mg/L glycine, and vitamins: 0.05 mg/L folic acid, 0.5 mg/L thiamine, 0.5 mg/L nicotinic acid, 0.5 mg/L pyridoxine, and 0.05 mg/L biotin. A pollen grain was considered viable if it produced a tube longer than the diameter of the grain (Roberts et al., 1983 ). To determine the statistical difference in pollen fertility/sterility between the two species, a standard t test was carried out using the Sigma Stat package (Jandel Scientific Software, Chicago, USA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
Inflorescences of both A. fourcroydes and A. angustifolia bloom from July to November. Late flowering, though rarely, can occur throughout the year (Table 1). It is difficult to find well-developed inflorescences of henequén because farmers cut the scape as soon as it starts developing to prevent the leaves from drying (Figs. 1, 2). The inflorescences of henequén are large (3–4 m tall) and covered with numerous bracteal leaves. Flowers from in umbellate clusters (i.e., lateral racemes), starting from the lowest branches. Flowering continues for several weeks from the base upwards, so that after the first few days the inflorescence presents flowers in all stages of development (Figs. 3, 4). Flowers are bisexual (hermaphrodite) with both pistil and stamens in the same flower. The morphology of the flowers is very similar in both species, but some clones of A. angustifolia have darker anthers than A. fourcroydes (Figs. 5, 6). The flowers are epigyneous, with petals, stamens, and anthers located above the ovary (Figs. 7, 8). The six narrow stamens are united at the base of the tube of the corolla. Their basal parts are fused with the pistil wall and form part of the ovary, a characteristic that is common to all agave species. The flowers are protandrous; the anthers (the androecium) are supported by long filaments and protrude 3–4 cm from the corolla (Figs. 7, 8). Flower development of this species is initiated by periclinal cell divisions in the protoderm (a meristem covered by a bract) where flower primordia (specific organ meristem layers) are formed. Six primordial stamens are initiated in the third whorl after the sepal and petal primordia, but prior to the initiation of carpel formation. At this stage the development of the gametophyte is initiated.



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  		Figs. 1–4. Inflorescences in Agave. 1. Scape development of A. fourcroydes is arrested after the leaves are cut. 2. Scapes cut by growers in commercial plantations of henequén. 3. Flowering inflorescence of A. angustifolia. 4. Flowering inflorescence of A. fourcroydes

 


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  		Figs. 5–8. Flowers of A. angustifolia and A. fourcroydes. 5. Flowering umbellate cluster of A. angustifolia. 6. Flowering umbellate cluster of A. fourcroydes. 7. Flowers of A. fourcroydes with unopened and opened (1st d) flowers. 8. Two stages of opened flowers (2nd and 3rd d) of A. angustifolia. Figure abbreviations: A, anthers; B, bracteole; F, filament; O, ovary body; P, pedicel; Pt, petal; S, style; St, stigma

 
Developmental stages of anthers and male gametophytic cells
Androecia of A. angustifolia and A. fourcroydes consist of six anthers, and are arcuate, tetrasporangiate, and dithecal with laterally positioned microsporangia that open longitudinally (Fig. 9). The stages of microsporangial development are shown in Table 2. The male gametophytic cycle begins with the formation of the microsporangium in the stamen and consists of archesporial cells (Fig. 11). This tissue forms a primary parietal and sporogenous layer of cells as well as secondary parietal layers (Fig. 12), with specific middle wall layers: the tapetum and the endothecium. The tapetum, whose cells form from the endothecium, is glandular (secretory). The microsporocytes or pollen mother cells (PMC) form via mitotic cell division of sporogenous tissue and can be observed, compactly arranged and connected through plasmodesmata, at the center of the microsporangium (Fig. 13). During the early meiotic prophase, the original plasmodesmata disintegrate and the PMC become spherical (Fig. 15). The tapetum in both agaves is best developed during microspore formation (Figs. 15–17), but, as can be seen in Fig. 14, in some anthers of A. fourcroydes the tapetum and the generative tissue are completely destroyed before microsporogenesis is initiated and therefore microspores and pollen grains do not develop.



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  		Figs. 9–10. Two cross sections through the flower buds at an earlier developmental stage of anthers and carpels; bar = 100 µm. 9. Transverse section across a 14-mm flower bud with androecium (six anthers). 10. Transverse section across a 36-mm flower bud with three carpels showing six ovaries and transmitting tissue. Figure abbreviations: A, anthers; C, carpel; Ov, ovaries; Pc, placental cells at the base of ovaries; Pt, petals; S, style; Sc, stylar canal with three grooves; Ss, stigmatic surface; Tt, transmitting tissue; Vt, vascular trace

 

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  		Table 2. Description of anther and ovary differentiation stages observed from histological analysis of agave flower buds

 


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  		Figs. 11–18. Microsporogenesis in Agave: transverse sections of methacrylate embedded anthers at different stages. 11. A 4-mm flower bud with differentiation of archesporial tissue; bar = 75 µm. 12. A 6-mm flower bud with sporogenious tissue and anther wall development; bar = 37.5 µm. 13. A 10-mm flower bud with microsporocytes and tapetum development; bar = 37.5 µm. 14. An 18-mm flower bud with destruction of tapetum and sporogenous tissue; bar = 75 µm. 15. An 18-mm flower bud with first meiotic division in microsporocytes; bar = 37.5 µm. 16. A 32-mm flower bud with formation of microspore tetrads; bar = 37.5 µm. 17. A 34-mm flower bud with free microspores; bar = 37.5 µm. 18. A 48-mm flower bud with mature pollen grains; bar = 37.5 µm. Figure abbreviations: As, archesporial cells; Ap, abortive pollen; Bp, binucleate pollen; C, connective; Cw, callose wall; E, epidermis; En, endothecium; Fmd, first meiotic divison (profase 1); Gc, generative cell; Ien, inner entine; M, middle layers; Msp, microspores; Oex, outer exine; PMC, pollen mother cells; Ps, pollen sac; S, sporogenous tissue; T, tapetum; t, tetrad of microspores; Td, tapetum destruction; Vn, vegetative cell nucleus

 
Microsporogenesis in these agaves takes place in two successive meiotic divisions forming T-shaped microspore tetrads (Figs. 15, 16). The uninucleate microspores grow after they are released from the tetrads (Fig. 17) and divide asymmetrically resulting in two types of cells (vegetative and generative). After meiosis, the tapetum begins to break down and the pollen grains are released. The mature pollen wall is composed of two layers: an outer one of exine and an inner one of intine (Fig. 18).

Pollen morphology and fertility
The morphology of the pollen of A. fourcroydes is very similar to that of A. angustifolia. The pollen grains are binuclear with vegetative and generative nuclei, monosulcate with two distal pores, and have a reticulate exine (Figs. 19, 20). A few differences between the two species can be observed in the pollen ornamentation and apertures (Ludlow-Wiechers and Ojeda, 1983 ; Ojeda, Ludlow, and Orellana, 1984 ). The proportion of spherical, mature pollen grains ("good pollen") differs between the two species: ~48.9 ± 1.5% for A. angustifolia and ~33.6 ± 0.08% for A. fourcroydes (Figs. 19, 20). This difference is statistically significant (t = 8.98 with 4.00 degrees of freedom at P ≤ 0.005, 95% confidence interval). The sterility of the pollen grains was also different: a very high frequency (66.4 ± 0.8%) of abnormal pollen grains was observed in henequén (Fig. 18) and 51.1 ± 1.5% for A. angustifolia. The difference between means is statistically significant (t = 6.65 with 2.00 degrees of freedom at P ≤ 0.022, 95% confidence interval). The percentage of germinated pollens that produced only short pollen tubes was very low (0.5 ± 0.08%). Occasionally, it was observed that in media 2, adjusted with vitamins, there was a slight increase in germination (1%) and in the length of the pollen tubes (Figs. 21, 22). On the other hand, pollen viability in A. angustifolia was ~3 ± 0.8% but the difference was not statistically significant. During germination the generative cells formed two sperm cells after another mitotic division (Fig. 22).



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  		Figs. 19–22. Mature agave pollen grains and in vitro germination. 19. "Good" pollen grains of A. angustifolia stained with acetocarmine; bar = 75 µm. 20. "Good" pollen grains of A. fourcroydes stained with acetocarmine; bar = 37.5 µm. 21. Pollen tube formation in A. fourcroydes after 4 h of in vitro germination in medium 1, bar = 37.5 µm. 22. Pollen tube formation in A. fourcroydes after 4 h of in vitro germination in medium 2; bar = 75 µm. Figure abbreviations: Ien, inner entine; Oex, reticulate outer exine; Dp, distal pore; Sc, sperm cells

 
Ovule and embryo sac formation
The gynoecium of A. fourcroydes and A. angustifolia consists of three united carpels and a trilocular ovary with axillary placentation (Fig. 10). Each locule has many anatropous ovules. The stages of the ovary differentiation process that are inferred from histological analyses are presented in Table 2. The ovules are specialized structures, derived from the placenta of the ovary, that produce the megasporocyte (Fig. 23). The latter is known as the megaspore mother cell (MMC) and is the site of the embryo sac formation, fertilization, and embryogenesis. The placenta is confined to the central axis of the pistil. The ovules are connected to the ovary wall that consists of three basic structures: a nucellus or megasporangium, two integuments (inner and outer), and a funiculus (Figs. 23, 24). The MMC, which forms from the archesporial tissue, is located below the apex of the nucellus (Fig. 24) while the integuments form at its base during megasporogenesis (Figs. 23, 24). The inner integument is located at the nucellus, whereas the outer integument is located above the inner integument and consists of two cell layers (Figs. 25, 26). The micropyle pore is found at the top of the outer integument (Fig. 26). The funiculus is fused with one side of the ovule and is visible as a rib (chalazal region) and located at the opposite end of the micropyle (Fig. 26).



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  		Figs. 23–31. Megasporogenesis in Agave: longisections of ovaries and ovules at different stages of development. 23. A 26-mm flower bud with ovary primordia; bar = 75 µm. 24. A 25-mm flower bud with young ovule showing the megasporocyte (MMC), bar = 37.5 µm. 25. A 36-mm flower bud with young ovule showing the first meiotic division of the megasporocyte with chromosomal aberrations; bar = 37.5 µm. 26. A 36-mm flower bud with young ovule showing two diad nuclei after the first meiotic division of the megasporocyte; bar = 37.5 µm. 27. A 38-mm flower bud, showing megaspores after the second meiotic division; bar = 37.5 µm. 28. A 38-mm flower bud, showing formation of embryo sac from megaspores; bar = 37.5 µm. 29. A 48-mm flower bud, showing embryo sac with cell migration; bar = 37.5 µm. 30. A 48-mm flower bud, tangential section through the embryo sac with polar nuclei of the central cell; bar = 37.5 µm. 31. A 48-mm flower bud, showing formed embryo sac; bar = 37.5 µm. Figure abbreviations: Ac, antipodal cells; Cf, chromosomal fragments; Cr, chalazal region; Ct, conducting tissue; Ec, egg cell; Es, embryo sac; Fu, funiculus; Ii, inner integument; Md, first meiotic division; Me, megasporocyte; Mi, micropile; Mt, megaspore tetrads; Nu, nucellus; Oi, outer integument; Pn, polar nuclei of the central cell

 
During megasporogenesis the MMC undergoes a meiotic division parallel to the micropylar-chalasal axis of the nucellus. At this stage, we observed some structures that could indicate that chromosomal fragmentations occur (Fig. 25). Wall formation is perpendicular to the micropylar-chalasal axis, forming a first dyad of megaspores (Fig. 26). Only one megaspore undergoes a second meiotic division creating two functional megaspores (bisporic type of megasporogenesis, Fig. 27). Thus the female gametophyte in henequén develops from the two viable micropile megaspores directly into an embryo sac (Fig. 28). However, their formation is not regular for a normal eight-nucleate megagametophyte. Positioned at the center of the embryo sac, the central cell contains two polar nuclei (Figs. 29, 30) that partially fuse before fertilization, generating the primary endosperm. The egg cell is located in the micropylar part of the embryo sac, while the synergids, which could be located on either side of the egg cell region, are not present in the mature embryo sac (Figs. 29, 31). The antipodal cells are placed at the chalazal region (Fig. 31).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
Henequén (Agave fourcroydes Lem.) is an economically important fiber crop for the state of Yucatán, México (Cruz, Orellana, and Robert, 1985; Colunga-García Marín and May-Pat, 1993) . It is, however, a relatively infertile pentaploid species with a very long life span, which is vegetatively propagated and has never been genetically improved. We studied the reproductive biology of this species in order to understand the basis of its sexual reproductive patterns and to predict the difficulties involved in a crossbreeding program. We decided to include its putative wild ancestor A. angustifolia in this study because, being phylogenetically related to the former, it could provide clues to its reproductive characteristics. A limitation of the present study was posed by the difficulty of finding enough flowering plants, particularly of henequén, and some of the variations observed could be due to the different environmental conditions under which the plants were growing.

Both species have the same vegetative and sexual reproductive strategies and are remarkably similar at the morphological and biochemical levels (Colunga-García Marín and May-Pat, 1997 ; Colunga-García Marín et al., 1999 ). However, A. angustifolia is a hexaploid species that produces a high percentage of fertile seeds. It is interesting to note that there is very little information regarding the development of the reproductive system of the Agavaceae (Wunderlich, 1950 ; Gentry, 1982 ), and these studies are limited to some aspects of pollination (Arizaga et al., 2000 ; Slauson, 2000) , nectar production, and seed viability (Schaffer and Schaffer, 1977, 1979 ; Freeman and Reid, 1985 ), while little is known about other aspects such as gametogenesis. In this paper we discuss the stages of anther and ovary development in henequén related to flower growth, compare them to those of A. angustifolia, and discuss the possible causes of the low fertility of the former.

Principal stages of gametophyte development
Flower development and maturation begin at the lowermost buds in the inflorescence and proceed upwards. The principal stages of gametophyte development are described in Table 2 and show the correlation of anther ontogeny and ovary differentiation with flower bud length. Both processes may be divided into three phases. Phase I (premeiosis) encompasses the early proliferative stages, the differentiation of the locules as well as the establishment of the PMC, and the formation of MMC. Phase II (meiotic stage) extends from the onset of the PMC and MMC meiosis to the appearance of the microspores and functional megaspores. Phase III (postmeiosis) begins at microspore mitosis and extends to the maturation of pollen grains of the male gametophyte and the formation of the embryo sac.

The results in Table 2 show that some important events of male and female gametogenesis are asynchronous. Microsporogenesis starts before megasporogenesis and, as a result, pollen maturity and anther dehiscence occur before the stigma becomes receptive. This pattern (protandria) is similar for both A. fourcroydes and A. angustifolia and has been described for other species such as A. sisalana (Lock, 1962 ) and A. lechugilla (Freeman and Reid, 1985 ). This asynchrony of gametophyte development might be a common pattern for the genus Agave. Self-pollination is therefore unlikely within the same flower, but mature pollen could fall on receptive flowers developing on lower parts of the scape.

Male gametophyte development
Microsporogenesis in A. angustifolia and A. fourcroydes with the "successive type" of meiosis was recognized because cell walls formed after the first cell division (Fig. 16). Although most stages of the process of male gamete formation in both agaves are apparently normal, pollen grains showed a very low in vitro germination rate: 3% for A. angustifolia and 0.5% for A. fourcroydes. This could reflect some meiotic and mitotic aberrations during microsporogenesis, due to their polyploid nature. Alternatively, it could be due to the influence of tapetal malfunction (Fig. 14), since it is widely assumed that the tapetum plays an important role during microsporogenesis and the maturation of the pollen grains (Chapman, 1987 ).

Although in vitro germination cannot reproduce exact in vivo conditions (Roberts et al., 1983 ), we believe it can be a useful tool to relate pollen quality (viability) to normal male gametophyte development, as previously discussed by Janssen and Hermsen (1976) .

Female gametophyte: ovule and embryo sac development
According to Dahlgren, Clifford, and Yeo (1985) , the female gametophyte of the Agavoideae is generated from the only functional megaspore via megagametogenesis (Polygonum type). However, in A. fourcroydes and A. angustifolia, we found a bisporic type of megasporogenesis (similar to Allium) but not the Polygonum type. The female gametophyte in henequén develops directly into an embryo sac from the two viable megaspores. These megaspores divide mitotically twice resulting in the migration of the nuclei and in the maturation of the embryo sac. Failures in meiosis might have resulted in the formation of abortive megaspores with a distorted chromosome balance that lead to the degradation of the nuclei and the occurrence of numerous empty embryo sacs (Fig. 25). It is apparent from the anatomy of the carpels and ovaries that structural maturation of the embryo sacs is not regular in some parts of the locules, and this could be the reason for the low fertility of the female gametophyte.

The results so far show that the formation of male and female gametophyte in henequén is abnormal at the meiotic and postmeiotic stages and that these alterations might be responsible for the low fertility shown by this species. These results should be useful for future henequén breeding research using pollination, fertilization, and zygotic embryo rescue.



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  		Figs. 23–31. Continued

 

   FOOTNOTES
 
1 The authors wish to thank two anonymous referees and Dr. Amarella Eastmond for their thorough reviews of the manuscript and relevant comments, and Dr. Roger Orellana for allowing us to collect plant material from CICY's Botanical Garden. This research was partially supported by funds provided by SISIERRA-CONACYT (Mexico), grant 980608. Back

2 Author for correspondence and reprint requests (npiven{at}cicy.mx ). Back


   LITERATURE CITED
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Arizaga S. E. Ezcurra E. Peters F. Ramírez de Arellano E. Vega 2000 Pollination biology of Agave macrocantha (Agavaceae) in a Mexican tropical desert. I. Floral biology and pollination mechanisms. American Journal of Botany 87: 1004-1010[Abstract/Free Full Text]

Berlin G. P. J. P. Miksche 1976 Botanical microtechnique and cytochemistry. 3rd ed. Iowa State University Press, Ames, Iowa, USA

Castorena-Sanchez I. R. M. Escobedo A. Quiroz 1991 New cytotaxonomical determinants recognized in six taxa of Agave in the sections Rigidae and Sisalanae. Canadian Journal of Botany 69: 1257-1264

Chapman G. P. 1987 The tapetum. In K. L. Giles and J. Pracash [eds.], Pollen: cytology and development. International Review of Cytology 107: 111-125[CrossRef]

Colunga-GarcÍa Marin P. J. Coello-Coello L. E. Eguiarte D. Piñero 1999 Isozymatic variation and phylogenetic relationships between henequén (Agave fourcroydes) and its wild ancestor A. angustifolia (Agavaceae). American Journal of Botany 86: 115-123[Abstract/Free Full Text]

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