Acessibilidade / Reportar erro

Embryology and cytogenetics of Eupatorium pauciflorum and E. intermedium (Compositae)

Abstracts

The embryology of Eupatorium pauciflorum indicates diplospory with autonomous endosperm development. The embryo sac is of the polygonum type and the polar nuclei mostly fuse before anthesis. The occurrence of precocious embryo and endosperm development in unopened florets, and the total absence of germinated pollen grains on exposed stigmas, as well as the absence of pollen tubes in the ovules, indicate agamospermy to be obligate and embryo and endosperm development autonomous. The study of microsporogenesis revealed the total absence of pollen production in consequence of microsporocyte degeneration before the onset of meiosis, which resulted in absolute male sterility. E. pauciflorum was demonstrated to be an autotriploid with a basic set of 10 chromosomes, each represented three times. Embryological studies showed E. intermedium to undergo reductive meiosis with tetrad formation during megasporogenesis, followed by monosporic embryo sac development of the polygonum type. The polar nuclei fuse before anthesis. The egg cell invariably attains anthesis still undivided, without precocious embryony. Meiosis of microsporogenesis results in the regular formation of 10 bivalents and the subsequent stages of microsporogenesis are normal. Stigmatic loads indicate the regular occurrence of pollination with viable, functional grains. Karyotypic studies revealed a complement of 20 chromosomes separable into 10 pairs. It is concluded that E. pauciflorum, as represented by the material studied, is apomictic while E. intermedium is sexual.


Os estudos embriológicos indicam que Eupatorium pauciflorum apresenta diplosporia com desenvolvimento autônomo do endosperma. O saco embrionário é do tipo polygonum e os núcleos polares se fundem antes da antese. A ocorrência de embrionia precoce e desenvolvimento do endosperma em flores fechadas, e a total ausência de grãos de pólen germinados em estigmas expostos, bem como a ausência de tubos polínicos nos óvulos, indicam que agamospermia é obrigatória e o desenvolvimento do embrião e do endosperma é autônomo. O estudo da microsporogênese revelou completa ausência de produção de pólen, em razão da degeneração dos microsporócitos antes do início da meiose, o que resulta em absoluta esterilidade masculina. E. pauciflorum demonstrou ser autotriplóide com um conjunto básico de 10 cromossomos repetidos três vezes. Os estudos embriológicos mostraram que E. intermedium sofre meiose reducional, com formação de tétrades durante a megasporogênese, seguida pelo desenvolvimento monospórico do saco embrionário do tipo polygonum. Os núcleos polares se fundem antes da antese. A oosfera invariavelmente permanece até a antese sem se dividir, impedindo a ocorrência de embrionia precoce. A meiose da microsporogênese resulta na formação regular de 10 bivalentes; os estágios subseqüentes da microsporogênese são normais e a análise da superfície estigmática indica a ocorrência regular de polinização, com grãos de pólen viáveis e funcionais. Estudos de cariótipo revelaram um complemento de 20 cromossomos separados em 10 pares. É possível concluir que E. pauciflorum, como representada pelo material estudado, é apomítica, enquanto E. intermedium é sexuada.


Embryology and cytogenetics of Eupatorium pauciflorum and E. intermedium (Compositae)

Maristela Sanches Bertasso-Borges1,2 and James Robert Coleman2

1Departamento de Ciências Biológicas, Centro Universitário de Rio Preto, UNIRP, Rua Ivete Gabriel Atique, 45, 15025-400 São José do Rio Preto, SP, Brasil. Send correspondence to M.S.B.-B. E-mail: bertasso@unirpnet.com.br

2Departamento de Botânica, Instituto de Biociências, Letras e Ciências Exatas de São José do Rio Preto, São José do Rio Preto, SP.

ABSTRACT

The embryology of Eupatorium pauciflorum indicates diplospory with autonomous endosperm development. The embryo sac is of the polygonum type and the polar nuclei mostly fuse before anthesis. The occurrence of precocious embryo and endosperm development in unopened florets, and the total absence of germinated pollen grains on exposed stigmas, as well as the absence of pollen tubes in the ovules, indicate agamospermy to be obligate and embryo and endosperm development autonomous. The study of microsporogenesis revealed the total absence of pollen production in consequence of microsporocyte degeneration before the onset of meiosis, which resulted in absolute male sterility. E. pauciflorum was demonstrated to be an autotriploid with a basic set of 10 chromosomes, each represented three times. Embryological studies showed E. intermedium to undergo reductive meiosis with tetrad formation during megasporogenesis, followed by monosporic embryo sac development of the polygonum type. The polar nuclei fuse before anthesis. The egg cell invariably attains anthesis still undivided, without precocious embryony. Meiosis of microsporogenesis results in the regular formation of 10 bivalents and the subsequent stages of microsporogenesis are normal. Stigmatic loads indicate the regular occurrence of pollination with viable, functional grains. Karyotypic studies revealed a complement of 20 chromosomes separable into 10 pairs. It is concluded that E. pauciflorum, as represented by the material studied, is apomictic while E. intermedium is sexual.

INTRODUCTION

Apomixis, or asexual reproduction through seeds, refers to a method of reproduction in which the embryo develops from an unfertilized egg and results in a progeny genetically identical to the mother plant. Thus used, apomixis is synonymous with agamospermy. Apomixis is divided on the basis of the occurrence or not of the megagametophytic generation. The formation of embryos directly from the nucellus or the integument of the ovule, without the occurrence of the megagametophytic generation, is referred to as adventitious embryony or sporophytic apomixis (Nogler, 1984; Koltunow, 1993; Khush et al., 1994). Gametophytic apomixis is subdivided in diplospory and apospory. In diplospory, the embryo sac arises from the megaspore mother cell by mitosis or modified meiosis, whereas in apospory the embryo sac arises from nucellar cells. In both cases, the resulting embryo sac is unreduced and embryo initiation is parthenogenetic (Nogler, 1984; Koltunow et al, 1995). Apomixis may be obligate or facultative, depending on whether seed production is entirely asexual or in part sexual. Embryo and endosperm initiation may be completely independent of pollination, resulting in autonomous development, or pollination may be necessary for fertilization of the central nucleus and endosperm initiation, phenomenon known as pseudogamy (Richards, 1986; Bashaw and Hanna, 1990).

Apomixis has been registered in relatively few families, among which the Compositae are prominent. Czapik (1996) affirmed that of 1535 genera pertaining to this family, 44 (2.9%) have reports of apomixis. In the genus Eupatorium, the first study of apomixis was done by Holmgren (1919) in E. glandulosum (= Ageratina adenophora, according to King and Robinson, 1987). Other studies have documented apomixis for some northern hemisphere species (Fryxell, 1957; Sparvoli, 1960; Sullivan, 1976), and more recently, apomixis has been demonstrated for South American species of this genus, one Argentine (Rozenblum et al., 1988) and four Brazilian (Coleman and Coleman, 1984, 1988; Coleman, 1989). Such studies indicate apomixis to be an important reproductive phenomenon in several species of the genus and a key to a better understanding of the evolution and systematics of the genus. The objective of the present study is to elucidate the embryology and cytogenetics of two additional Brazilian species, E. pauciflorum HBK (= Praxelis pauciflora (HBK) King and Rob. and E. intermedium DC (= Grazielia intermedia (DC) King and Rob.).

MATERIAL AND METHODS

The material of both species was collected from natural populations. Eupatorium pauciflorum was collected in the city of São José do Rio Preto and E. intermedium in the city of São Paulo (Parque do Estado de São Paulo), both in the State of São Paulo, Brazil. Voucher specimens are deposited in the herbarium of the Instituto de Botânica de São Paulo (SP) and in the herbarium of the Instituto de Biociências, Letras e Ciências Exatas de São José do Rio Preto (SJRP). Buds for embryological studies were fixed in FAA. Ovules were dissected from the ovaries, cleared in 4 ½ clearing solution (Herr Jr., 1971, 1972) and mounted directly in Hoyer's medium (Alexopoulos and Benke, 1952). Buds for the study of microsporogenesis were fixed in 1:3 acetic acid-ethanol. Staining was done in acetocarmine and slides were made permanent with Hoyer's medium. Karyotypic studies were done using root tips of seedlings obtained by germinating achenes on humid filter paper in Petri dishes. The root tips were pre-treated in 0.002 M 8-hydroxyquinoline for 6 h, at 15 ± 3°C, and fixed in 1:3 acetic acid-ethanol. Hydrolysis was done in 1 N HCl for 5 min at 60°C. After staining with acetocarmine, the apices of the root tips were squashed in Hoyer's medium. Selected cells were photographed and prints for analysis made at 3750X. Metaphase chromosomes of one cell of E. pauciflorum and six cells of E. intermedium were measured. A Zeiss Standard WL photomicroscope equipped with phase constrast was used for all work. Germination tests were made on humid filter paper in Petri dishes, under ambiental conditions, and in a germination chamber, under conditions of constant temperature (30°C) and continuous light.

RESULTS AND DISCUSSION

E. pauciflorum is an annual, weedy plant of frequent occurrence in southern to central Brazil (Lorenzi, 1991). The plants produce numerous achenes. An analysis of 1650 achenes from 10 plants revealed that 84.8% (1399) contained an embryo. Values for individual plants ranged from 77.5 to 93.1%. Germination tests (50 achenes from each of 10 plants) were made in a germination chamber and under ambiental conditions. In the first case 5% (25) of the achenes germinated, values for individual plants varying from 2 to 8%, whereas under ambiental conditions 33.4% (167) germinated, values for individual plants varying from 10 to 54%.

An analysis of 1938 ovules in the initial stages of development revealed the total absence of megaspore formation. The megasporocyte (Figure 1a) was observed to serve directly as a megaspore, its nucleus dividing to give origin to a two-nucleate embryo sac (Figure 1b) whose nuclei migrate to opposite ends of the cell. A second division produces a four-nucleate embryo sac (Figure 1c), and a third division results in the formation of an unreduced eight-nucleate embryo sac, containing two synergids, egg cell, two polar nuclei (Figure 1d) and three antipodals. The polar nuclei fuse to form the central nucleus and the synergids degenerate. The egg cell and the central nucleus divide parthenogenetically to initiate the embryo and endosperm, respectively (Figure 1e,f).

Figure 1
- Embryo sac and embryo formation in Eupatorium pauciflorum. a, Megasporocyte; b, 2-nucleate embryo sac; c, 4-nucleate embryo sac; d, embryo sac showing one synergid, egg and polar nuclei; e, embryo sac with egg and central nucleus; f, embryo sac with embryo and endosperm. a-f, 780X.

The affirmation that the megaspore nucleus divides mitotically is based on the observation that no ovule showed dyads or tetrads of megaspores, which indicates the lack or modification of meiosis. The embryo sac results from three successive divisions of the megasporocyte, which permits the conclusion that embryo sac formation in E. pauciflorum is diplosporic. Diplospory has been reported for E. glandulosum (Holmgren, 1919), E. callilepis (Coleman and Coleman, 1984), E. squalidum (Coleman and Coleman, 1988) and E. odoratum (Coleman, 1989). Among the Brazilian species of the genus, apospory has been observed only in E. bupleurifolium (Coleman and Coleman, 1984).

Florets were considered to be in pre-anthesis when judged to be within 24 h of anthesis. In pre-anthesis 55.9% of the ovules already contained an embryo (Table I). The determination of precocious embryony confirms agamospermy in the material studied. Observations made of ovules in anthesis showed an egg cell remaining in only 6% of the cases (Table I), which demonstrates that in E. pauciflorum precocious embryony represents a strong barrier to sexual reproduction. No ovule showed the presence of a pollen tube. The presence of endosperm in ovules of florets in pre-anthesis (42.41%) indicates autonomous development (Table I). Nogler (1984) affirmed that autonomous endosperm development is typical for apomictic Compositae. It has also been reported for E. glandulosum (Holmgren, 1919), E. bupleurifolium and E. callilepis (Coleman and Coleman, 1984), E. odoratum (Coleman, 1989) and E. tanacetifolium (Rozenblum et al., 1988). Embryo formation in E. pauciflorum invariably precedes endosperm formation (Table I). Vijayaraghavan and Prabhakar (1984) affirmed that this phenomenon is of physiological interest because it provides irrefutable evidence to support the position that early embryo development is not sustained by nutrients of the endosperm but by nutrients transferred to the embryo sac from surrounding tissues.

The study of microsporogenesis in E. pauciflorum revealed the total absence of pollen production in consequence of microsporocyte degeneration before the onset of meiosis which resulted in absolute male sterility in the material studied. The total failure of pollen production results in total absence of pollination. This was confirmed since only two of 125 pistils examined from five plants had a single pollen grain. The two grains observed probably came from a different species. Low or no pollen production and precocious embryony indicate sexual reproduction to be very rare or absent in the material studied. Male sterility has also been demonstrated for diplosporic E. odoratum (Coleman, 1989).

The results of the karyotypic study, although based on a single cell, suggest the presence of a basic set of 10 chromosomes repeated three times (Figure 2), which would characterize the species as an autotriploid. Mean chromosome length ranged from 1.07 to 1.74 µm (Table II). The additional use of arm ratio was essential for the identification of homologs because of the gradual variation in chromosome length. All the chromosomes were submetacentric following the terminology of Levan et al. (1964). No SAT-chromosomes were observed. It is probable that the autotriploid origin of E. pauciflorum occurred by the union of a reduced with an unreduced gamete in a sexual, diploid population of the species. E. squalidum (Coleman and Coleman, 1988) and E. bupleurifolium (Coleman and Coleman, 1984) are also of autotriploid origin. A strong association between gametophytic apomixis and polyploidy is well known. Richards (1996) affirmed that this association is explained through the expression in the megagametophyte of recessive lethal genes linked to genes for apomixis. Many of these lethals would be expressed in a haploid embryo sac, but not in the unreduced embryo sac of the gametophytic apomict, where they are protected from expression in a heterozygous condition. Such genes would also be expressed in haploid pollen tubes, turning endosperm initiation in diploid pseudogamous apomicts impossible. This model also affirms that sporophytic apomictics are frequently diploids because this mechanism of agamospermy does not involve a gametophytic generation and thus the lethal recessive genes are not expressed.

Figure 2
- Mitotic chromosomes in Eupatorium pauciflorum. a, Mitotic metaphase showing 30 chromosomes; b, karyotype indicating the presence of 10 basic chromosomes, each present in triplicate; c, ideogram. In a and b, bar represents 5 µm.

Eupatorium intermedium is a subshrub of southern to central Brazil which occurs in stable environments. An analysis of 1730 achenes from seven plants revealed that 2.8% contained an embryo. Values for individual plants ranged from 0 to 5.4%. In germination tests (60 achenes) done in a germination chamber 41 of the achenes germinated.

Embryological studies showed that the megasporocyte (Figure 3a) underwent a first meiotic division to form a dyad (Figure 3b) which rapidly underwent a second meiotic division to produce a tetrad of reduced megaspores (Figure 3c). The presence of dyads and tetrads indicates that reduction is a regular feature of megasporogenesis in this species (Table III). The chalazal megaspore enlarges to form the one-nucleate embryo sac (Figure 3d), which crushes the three micropylar megaspores. The embryo sac passes through the two-nucleate (Figure 3e) and four-nucleate stages (Figure 3f), resulting in a reduced eight-nucleate embryo sac composed of egg cell, two polar nuclei (Figure 4a), two synergids and three antipodals. Embryo sac formation is, thus, of the polygonum type, which is apparently typical for the genus (Davis, 1966).

Figure 3
- Embryo sac formation in Eupatorium intermedium. a, Megasporocyte; b, dyad; c, tetrad of megaspores; d, 1-nucleate embryo sac; e, 2-nucleate embryo sac; f, 4-nucleate embryo sac. a-f, 810X.
Figure 4
- Mature embryo sac structure and embryo formation in Eupatorium intermedium. a, Embryo sac showing egg and polar nuclei; b, embryo sac with egg and central nucleus; c, embryo sac with initial divisions of embryo and endosperm; d, embryo (globular fase) and endosperm. a, 635X; b, 900X; c, 450X; d, 430X.

The polar nuclei fuse to form the central nucleus (Figure 4b), usually before anthesis (Table III). The egg cell invariably attains anthesis still undivided, precocious embryony not occurring. The low frequency of embryo and central nucleus (0.72%) at anthesis indicates the embryo and endosperm (Figure 4c,d) initiate nearly simultaneously. Similar results were observed for E. inulaefolium (Coleman, 1991).

The analysis of chromosome pairing at diakinesis revealed the uniform occurrence of 10 bivalents (Figure 5a). Homologs separate normally, resulting in anaphase I with 10 chromosomes at each pole (Figure 5b). The regularity of microsporogenesis, confirmed by the presence of normal tetrads of microspores (Figure 5c) and good pollen formation, indicates a high degree of male fertility for the material studied, which is in accordance with its sexual nature. Counts of pollen grains on 90 stigmas of three plants showed that 67 carried an average of 4.4 germinated grains, indicating the regular occurrence of pollination with viable, functional grains.

Figure 5
- Microsporogenesis in Eupatorium intermedium. a, Diakinesis showing 10 bivalents; b, anaphase I with 10 chromosomes at each pole; c, normal tetrads of microspores. a, 1725X; b, c, 1700X.

Karyotype analysis of six cells showed 20 chromosomes which could be separated into 10 pairs, indicating the chromosome complement to be composed of a basic set of 10 chromosomes repeated twice (Figure 6a,b). Table IV presents the mean measurements for the 10 chromosomes of the basic set and Figure 6c presents the ideogram. Mean chromosome length ranged from 2.66 to 4.07 µm (Table IV). All chromosomes were submetacentric. Only one cell showed the presence of SAT-chromosomes. The satellites apparently occurred on the chromosome pair with the highest arm ratio (2.06), which is also the shortest of the complement, corresponding to chromosome 10 (Figure 6).

Figure 6
- Mitotic chromosomes in Eupatorium intermedium. a, Mitotic metaphase showing 20 chromosomes; b, karyotype indicating the presence of 10 basic chromosomes; c, ideogram. In a and b, bar represents 5 µm.

We conclude that E. intermedium is sexual while E. pauciflorum is apomictic. The evidence that sustains this conclusion for E. pauciflorum is: absence of reductional meiosis during megasporogenesis, precocious formation of embryo and endosperm, total male sterility, absence of pollination and the triploid nature of the species.

ACKNOWLEDGMENTS

We thank Maria Helena Carabolante for technical assistance. This work was supported by CAPES. Publication supported by FAPESP.

RESUMO

Os estudos embriológicos indicam que Eupatorium pauciflorum apresenta diplosporia com desenvolvimento autônomo do endosperma. O saco embrionário é do tipo polygonum e os núcleos polares se fundem antes da antese. A ocorrência de embrionia precoce e desenvolvimento do endosperma em flores fechadas, e a total ausência de grãos de pólen germinados em estigmas expostos, bem como a ausência de tubos polínicos nos óvulos, indicam que agamospermia é obrigatória e o desenvolvimento do embrião e do endosperma é autônomo. O estudo da microsporogênese revelou completa ausência de produção de pólen, em razão da degeneração dos microsporócitos antes do início da meiose, o que resulta em absoluta esterilidade masculina. E. pauciflorum demonstrou ser autotriplóide com um conjunto básico de 10 cromossomos repetidos três vezes. Os estudos embriológicos mostraram que E. intermedium sofre meiose reducional, com formação de tétrades durante a megasporogênese, seguida pelo desenvolvimento monospórico do saco embrionário do tipo polygonum. Os núcleos polares se fundem antes da antese. A oosfera invariavelmente permanece até a antese sem se dividir, impedindo a ocorrência de embrionia precoce. A meiose da microsporogênese resulta na formação regular de 10 bivalentes; os estágios subseqüentes da microsporogênese são normais e a análise da superfície estigmática indica a ocorrência regular de polinização, com grãos de pólen viáveis e funcionais. Estudos de cariótipo revelaram um complemento de 20 cromossomos separados em 10 pares. É possível concluir que E. pauciflorum, como representada pelo material estudado, é apomítica, enquanto E. intermedium é sexuada.

REFERENCES

Alexopoulos, C.J. and Benke, E.S. (1952). Laboratory Manual for Introductory Mycology. Burgess Publishing Co., Minneapolis.

Bashaw, E.C. and Hanna, W.W. (1990). Apomictic reproduction. In: Reproductive Versatility in the Grasses (Chapman, G.P., ed.). Cambridge University Press, London, pp. 100-130.

Coleman, J.R. (1989). Embryology and cytogenetics of apomictic hexaploid Eupatorium odoratum L. (Compositae). Rev. Bras. Genet.12: 803-817.

Coleman, J.R. (1991). Embryology and cytogenetics of sexual tetraploid Eupatorium inulaefolium H.B.K. Rev. Bras. Genet. 14: 123-133.

Coleman, J.R. and Coleman, M.A. (1984). Apomixis in two triploid Brazilian species of Eupatorium: E. bupleurifolium and E. callilepis. Rev. Bras. Genet. 7: 549-567.

Coleman, J.R. and Coleman, M.A. (1988). Embryology and cytogenetics of apomictic triploid Eupatorium squalidum DC (Compositae). Rev. Bras. Genet. 11: 129-148.

Czapick, R. (1996). Frequency of agamospermy. http://cimmyt/biotechnology/apomixis/newsletter/9/ANL 9-8.htm.

Davis, G.L. (1966). Systematic Embryology of the Angiosperms. John Wiley & Sons, Inc., New York.

Fryxell, P.A. (1957). Mode of reproduction of higher plants. Bot. Revi. 23: 135-233.

Herr Jr., J.M. (1971). A new clearing squash technique for the study of ovule development in angiosperms. Am. J. Bot. 58: 785-790.

Herr Jr., J.M. (1972). Applications of a new clearing technique for the investigation of vascular plant morphology. J. Elisha Mitchell Sci. Soc. 88: 137-143.

Holmgren, I. (1919). Zytologische Studien über die Fortpflanzung bei den Gattungen Erigeron und Eupatorium. Kgl. Sv. Vet. Ak. Handl. 59: 1-118.

Khush, G.S., Brar, D.S., Bennett, J. and Virmani, S.S. (1994). Apomixis for rice improvement. In: Apomixis: Exploiting Hybrid Vigor in Rice (Khush, G.S., ed.). International Rice Research Institute, Philippines, pp. 1-21.

King, R.M. and Robinson, H. (1987). The Genera of the Eupatorieae (Asteraceae). Missouri Botanical Garden, Kansas.

Koltunow, A.M. (1993). Apomixis: embryo sacs and embryos formed without meiosis or fertilization in ovules. Plant Cell 5: 1425-1437.

Koltunow, A.M., Bicknell, R.A. and Chaudhury, A.M. (1995). Apomixis: molecular strategies for the generation of genetically identical seeds without fertilization. Plant Physiol. 108: 1345-1352.

Levan, A., Fredga, K. and Sandberg, A.A. (1964). Nomenclature for centromeric position on chromosomes. Hereditas 52: 201-220.

Lorenzi, H. (1991). Plantas Daninhas do Brasil: Terrestres, Aquáticas, Parasitas e Medicinais. Editora Plantarum Ltda., Nova Odessa.

Nogler, G.A. (1984). Gametophytic apomixis. In: Embryology of Angiosperms (Johri, B.M., ed.). Springer-Verlag, Berlin, Heidelberg, New York, pp. 475-517.

Richards, A.J. (1986). Plant Breeding Systems. George Allen and Unwin, London.

Richards, A.J. (1996). Why is gametophytic apomixis almost restricted to polyploids? The gametophyte-expressed lethal model. http://cimmyt/biotechnology/apomixis/newsletter/9/ANL 9-3.htm.

Rozenblum, E., Maldonado, S. and Waisman, C.E. (1988). Apomixis in Eupatorium tanacetifolium (Compositae). Am. J. Bot. 75: 311-322.

Sparvoli, E. (1960). Osservazioni cito-embriologiche in Eupatorium riparium. II. Megasporogenesi e sviluppo del gametofito femminile. Ann. Bot. 26: 481-504.

Sullivan, V.I. (1976). Diploidy, polyploidy and agamospermy among species of Eupatorium (Compositae). Can. J. Bot. 54: 2907-2917.

Vijayaraghavan, M.R. and Prabhakar, K. (1984). The endosperm. In: Embryology of Angiosperms (Johri, B.M. ed.). Springer-Verlag, Berlin, Heidelberg, New York, pp. 319-376.

(Received August 5, 1998)

  • Alexopoulos, C.J. and Benke, E.S. (1952). Laboratory Manual for Introductory Mycology. Burgess Publishing Co., Minneapolis.
  • Bashaw, E.C. and Hanna, W.W. (1990). Apomictic reproduction. In: Reproductive Versatility in the Grasses (Chapman, G.P., ed.). Cambridge University Press, London, pp. 100-130.
  • Coleman, J.R. (1989). Embryology and cytogenetics of apomictic hexaploid Eupatorium odoratum L. (Compositae). Rev. Bras. Genet.12: 803-817.
  • Coleman, J.R. (1991). Embryology and cytogenetics of sexual tetraploid Eupatorium inulaefolium H.B.K. Rev. Bras. Genet. 14: 123-133.
  • Coleman, J.R. and Coleman, M.A. (1984). Apomixis in two triploid Brazilian species of Eupatorium: E. bupleurifolium and E. callilepis Rev. Bras. Genet. 7: 549-567.
  • Coleman, J.R. and Coleman, M.A. (1988). Embryology and cytogenetics of apomictic triploid Eupatorium squalidum DC (Compositae). Rev. Bras. Genet. 11: 129-148.
  • Czapick, R. (1996). Frequency of agamospermy. http://cimmyt/biotechnology/apomixis/newsletter/9/ANL 9-8.htm.
  • Fryxell, P.A. (1957). Mode of reproduction of higher plants. Bot. Revi. 23: 135-233.
  • Herr Jr., J.M. (1971). A new clearing squash technique for the study of ovule development in angiosperms. Am. J. Bot. 58: 785-790.
  • Herr Jr., J.M. (1972). Applications of a new clearing technique for the investigation of vascular plant morphology. J. Elisha Mitchell Sci. Soc. 88: 137-143.
  • Holmgren, I. (1919). Zytologische Studien über die Fortpflanzung bei den Gattungen Erigeron und Eupatorium Kgl. Sv. Vet. Ak. Handl. 59: 1-118.
  • Khush, G.S., Brar, D.S., Bennett, J. and Virmani, S.S. (1994). Apomixis for rice improvement. In: Apomixis: Exploiting Hybrid Vigor in Rice (Khush, G.S., ed.). International Rice Research Institute, Philippines, pp. 1-21.
  • King, R.M. and Robinson, H. (1987). The Genera of the Eupatorieae (Asteraceae) Missouri Botanical Garden, Kansas.
  • Koltunow, A.M. (1993). Apomixis: embryo sacs and embryos formed without meiosis or fertilization in ovules. Plant Cell 5: 1425-1437.
  • Koltunow, A.M., Bicknell, R.A. and Chaudhury, A.M. (1995). Apomixis: molecular strategies for the generation of genetically identical seeds without fertilization. Plant Physiol. 108: 1345-1352.
  • Levan, A., Fredga, K. and Sandberg, A.A. (1964). Nomenclature for centromeric position on chromosomes. Hereditas 52: 201-220.
  • Lorenzi, H. (1991). Plantas Daninhas do Brasil: Terrestres, Aquáticas, Parasitas e Medicinais. Editora Plantarum Ltda., Nova Odessa.
  • Nogler, G.A. (1984). Gametophytic apomixis. In: Embryology of Angiosperms (Johri, B.M., ed.). Springer-Verlag, Berlin, Heidelberg, New York, pp. 475-517.
  • Richards, A.J. (1986). Plant Breeding Systems George Allen and Unwin, London.
  • Richards, A.J. (1996). Why is gametophytic apomixis almost restricted to polyploids? The gametophyte-expressed lethal model. http://cimmyt/biotechnology/apomixis/newsletter/9/ANL 9-3.htm.
  • Rozenblum, E., Maldonado, S. and Waisman, C.E. (1988). Apomixis in Eupatorium tanacetifolium (Compositae). Am. J. Bot. 75: 311-322.
  • Sparvoli, E. (1960). Osservazioni cito-embriologiche in Eupatorium riparium II. Megasporogenesi e sviluppo del gametofito femminile. Ann. Bot. 26: 481-504.
  • Sullivan, V.I. (1976). Diploidy, polyploidy and agamospermy among species of Eupatorium (Compositae). Can. J. Bot. 54: 2907-2917.
  • Vijayaraghavan, M.R. and Prabhakar, K. (1984). The endosperm. In: Embryology of Angiosperms (Johri, B.M. ed.). Springer-Verlag, Berlin, Heidelberg, New York, pp. 319-376.

Publication Dates

  • Publication in this collection
    01 Mar 1999
  • Date of issue
    Dec 1998

History

  • Received
    05 Aug 1998
Sociedade Brasileira de Genética Rua Cap. Adelmio Norberto da Silva, 736, 14025-670 Ribeirão Preto SP Brazil, Tel.: (55 16) 3911-4130 / Fax.: (55 16) 3621-3552 - Ribeirão Preto - SP - Brazil
E-mail: editor@gmb.org.br