On-line version ISSN 1678-4685
Genet. Mol. Biol. vol. 21 n. 4 São Paulo Dec. 1998
Hermaphroditism in the rubber tree Hevea brasiliensis (Willd. ex Adr. de Juss.) Muell. Arg. - II
Silvia Marina Cuco and Gerhard Bandel
Departamento de Genética, Escola Superior de Agricultura Luiz de Queiroz, Campus Luiz de Queiroz, USP, Caixa Postal 83, 13400-970 Piracicaba, SP, Brasil. Send correspondence to S.M.C.
Flowers of three Hevea brasiliensis clones, RRIM 527, RRIM 600 and GT 1, were analyzed under stereomicroscope and scanning electron microscope, aiming to observe hermaphroditism rates. Results showed 71.49% hermaphrodite flowers, 29.83% of which exhibited incompletely developed, residual anthers. The scanning electron microscope analysis did not detect differences in anther epidermis of male and bisexual flowers of RRIM 600 and RRIM 527. In GT 1 clone (sterile male), the anther epidermis was already weak at the beginning of floral development and completely wrinkled at the end of maturation. Consequently, the anthers were empty by this stage.
Rubber tree, Hevea brasiliensis (Willd. ex Adr. de Juss.) Muell. Arg., is a monoecious species belonging to the Euphorbiaceae family, which has 11 Hevea species presently known (Schultes, 1977; Gonçalves et al., 1983). It is a very important species, because it is the principal source of commercial natural rubber. Latex is a high molecular weight polymer whose structure is cis-1,4-polysoprene, produced in a specialized system (latex vessels) present in all organs of the plant (Mathew, 1992).
References of Hevea morphology describe it as a monoecious plant with unisexual flowers arranged in the same inflorescence (Bouychou, 1963; Compagnon, 1986; Wycherley, 1992). Female flowers terminate the main and secondary branches in the panicle, and males occupy all the remaining positions. In large inflorescences, we can find nearly 3,000 male flowers with an approximate proportion of one female to 60 male flowers (Bouychou, 1963).
The female flower has a more bulky calyx basis and is a little larger than the male. It has a tricarpelar ovary, sessil stigma and each carpel delimits a locule generally containing just one ovule. Staminodes can be present in the ovary basis. The male flower has ten sessile stamens directly inserted in the staminal column, in two verticils of five stamens each (Gonçalves et al., 1983, 1989).
In spite of the references that describe the flowers as unisexual, evidence of hermaphroditism has been obtained along the years. Warmke (1950) observed that 50% of supposedly female flowers of one Hevea brasiliensis plant were bisexual, showing one to five little, functional stamens in the ovary basis. Bouharmont (1960) observed the occurrence of the same bisexual flowers, having fertile or no anthers, in H. collina but rarely in H. brasiliensis. A more recent study reported 25.81% hermaphrodite flowers in four H. brasiliensis clones (Cuco and Bandel, 1994).
Rubber tree is regarded as having alogamous insect pollination (Wycherley, 1992), but selfcrossing also occurs on various levels (Bouychou, 1963). Also, selfincompatible and sterile male clones are known (Gonçalves, 1986). Thus, occurrence of high rates of hermaphroditism can change the panorama of Hevea's reproductive biology and alter genetic breeding strategies.
With this in mind, the present study was conducted to evaluate the occurrence of hermaphroditism in this species. Flowers were also analyzed with regard to stamen morphology of two different floral types. Analysis was performed with a scanning electron microscope.
MATERIAL AND METHODS
Flower buds of three Hevea brasiliensis clones, RRIM 527, RRIM 600 and GT 1, were collected from mature trees (13 years old) from plantations at ESALQ/USP in Piracicaba/SP. The buds were fixed in ethanol-acetic acid 3:1 and mantained in 70% ethanol under refrigeration. Flowers with external female morphology were dissected and analyzed under stereomicroscope, with magnification of 60 to 160 times.
For the scanning electron microscope (SEM) analysis, fixed flowers were dehydrated through aceton series (30, 50, 70, 95 and 100%) for 10 min each. Subsequently, they were dried to a critical point, and metalized with 180 to 260 s of gold. Photomicrographs were made in SEM Zeiss DSM 940A, using Ilford FP4 plus 120 or Fuji Neopan SS 120 films.
RESULTS AND DISCUSSION
A total of 1,217 flowers with external female morphology was analyzed. Table I shows floral composition observed for the three clones.
We can see that hermaphroditism was high in the three clones. The bisexual flower had a normal ovary with stamens parcially or completely around its basis (Figure 1). These anthers are normally smaller than those in male flowers, though sometimes with normal appearance and pollen grains (Figure 2). It was observed that 29.83% hermaphrodite flowers had incompletely developed, residual anthers (staminodes) (Figure 3). We can see in Table I that RRIM 600 and GT 1 had a high number of staminodes, 68.36% and 95.27%, respectively. In the RRIM 527 clone only 7.6% hermaphrodite flowers showed staminodes; therefore, 92.40% were typically bisexual, with developed stamens. A low number of exclusively female flowers were observed in RRIM 527 and RRIM 600, 19.30% and 27.25%, respectively. In the sterile male GT 1 clone, 48.07% of flowers were exclusively female and of the remaining 51.93% hermaphrodites, 95.27% had residual stamens. These data corroborate a previous study (Cuco and Bandel, 1994) in which high rates of bisexual flowers were observed.
Figure 1 - A) Ovary of hermaphrodite flower of RRIM 527 clone; in its basis we observe one developed and two residual anthers. B) Ovary of RRIM 527 flower surrounded by developed stamens. C) A detail of the epidermis of these anthers. Bars correspond to 200 mm and 500 mm, respectively.
Figure 2 - A) Developed anthers in the ovary basis of hermaphrodite flower of RRIM 527. B) A detail of this anther showing pollen grains in the slit. Bars correspond to 200 mm and 50 mm, respectively.
Figure 3 - Ovary of RRIM 600 flower with residual anthers in the basis. Bar corresponds to 200 mm.
Scanning electron microscopy analysis was performed in order to verify morphological differences in stamens of male and hermaphrodite flowers. As illustrated in Figures 1B and 4, the anther epidermis in male and bisexual flowers were similar. In contrast, immature GT 1 flower buds had wrinkled epidermis and empty stamens at maturation (Figure 5), due to male-sterility in this clone. During collection we observed that, contrary to other clones, GT 1 flower buds released more easily from petiolous and handling or wind could knock them down. Figures 6 and 7 compare inflorescences of GT 1 and RRIM 600. In RRIM 600, the inflorescences are more compact, having great number of male and female or bisexual flowers. In GT 1, the inflorescences remain almost exclusively females/hermaphrodites.
Figure 4 - A) Stamens of male flower of RRIM 527 clone. A) Anther of hermaphrodite flower of RRIM 527. Note the similarity between epidermis. Bars correspond to 100 mm and 50 mm, respectively.
Figure 5 - Stamens of GT 1 male flower in the begining (A) and end (B) of development. Bars correspond to 100 mm and 50 mm, respectively.
Figure 6 - GT 1 inflorescence with female flowers at the central and secondary axis extremity and some males.
Figure 7 - RRIM 600 inflorescence with numerous female and male flowers.
With these results, we conclude that hermaphroditism rate is high in the three H. brasiliensis clones studied, with 71.49% bisexual flowers, with residual or developed anthers. No morphological differences at the epidermis level were detected between anthers of male and hermaphrodite flowers. Externally, it was impossible to distinguish the two floral types. In addition, the sterile male clone GT 1 already showed empty anthers during the initial development of flower buds, which is a physiologically coherent fact.
We are grateful to Prof. Dr. Elliot W. Kitajima, from NAP-MEPA/ESALQ-USP, for his suggestions. Research supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). Publication supported by FAPESP.
Flores de três clones de Hevea brasiliensis, RRIM 527, RRIM 600 e GT 1, foram analisadas, sob lupa e microscopia eletrônica de varredura, a fim de se observar as taxas de ocorrência de hermafroditismo. Os resultados mostraram um total de 71,49% de flores hermafroditas, sendo que destas 29,83% apresentaram anteras residuais, não completamente desenvolvidas. As análises ao microscópio de varredura não mostraram diferença ao nível de epiderme de anteras em flores masculinas e hermafroditas de RRIM 527 e RRIM 600. No clone GT 1 (macho estéril) a epiderme das anteras mostrou-se frouxa já no início do desenvolvimento floral e completamente enrugada ao final da maturação, demonstrando que as anteras estão vazias neste estádio.
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(Received September 5, 1997)