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a Dep. of Crop Sciences, Univ. of Illinois, 1102 South Goodwin Avenue, Urbana, IL 61801 USA
soyui{at}uiuc.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMaterials and methodsResults and discussionREFERENCES |
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Abbreviations: MMC, megaspore mother cell • PMC, pollen mother cell
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMaterials and methodsResults and discussionREFERENCES |
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Although autotriploids in soybean have not been produced from crosses between autotetraploids and diploids, a large number of triploid plants have been isolated from the progeny of different lines of homozygous recessive male-sterile (ms1ms1) soybean (Kenworthy et al., 1973; Cutter and Bingham, 1977; Beversdorf and Bingham, 1977; Chen et al., 1985; Chen and Palmer, 1985; Sorrells and Bingham, 1979). Autotriploids derived from ms1ms1 soybean always carry the recessive ms1 gene either in homozygous (ms1ms1ms1) or in heterozygous (Ms1ms1ms1) conditions and have poor seed set. Chen and Palmer (1985) obtained 138 plants with greater than 40 chromosomes (2n = 44 to 2n = 71), but not 41 chromosomes, from 32 male-fertile (Ms1ms1ms1) triploid plants. Therefore, the triploids from ms1ms1 soybean have not become useful sources for generating primary trisomics (2n = 2x + 1 = 41) in soybean.
Recently, we obtained a spontaneous hypertriploid (2n = 3x + 1 = 61) in the progeny of a marker stock T31 (glabrous, p2p2) and a primary trisomic line T190-47-3. The objectives of this study were to evaluate the origin, morphology, cytology, fertility, and breeding behavior of a hypertriploid.
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Materials and methods |
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TOPABSTRACTINTRODUCTIONMaterials and methodsResults and discussionREFERENCES |
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The hypertriploid and disomic sib plants were individually planted in 20-cm clay pots in the greenhouse. General morphologies of the hypertriploid and its disomic sib were compared during vegetative development. The length and width of 10 intermediate trifoliolate terminal leaflets were measured when plants were near senescence (Gwyn et al., 1985).
The young flower buds from the hypertriploid were sampled before flowering for meiotic studies (Singh and Hymowitz, 1988). Briefly, young flower buds were fixed in a mixture of 3:1 ethanol (95%, v/v)/propionic acid, with the addition of 1 g of ferric chloride per 100 mL. After 24 to 48 h of fixation, the buds were transferred to 70% (v/v) ethanol and stored in a refrigerator. Anthers with meiotic stages were stained in 1% (w/v) propiono-carmine for 1 wk and were squashed in 45% (v/v) acetic acid. Meiotic chromosome pairing configurations at Metaphase I and chromosome migration were analyzed only in microsporocytes with well-spread chromosomes.
At anthesis, pollen samples from the hypertriploid and its disomic sib were stained with Lugol solution (L-6146, Sigma Chemical Co., St. Louis, MO), and 500 pollen grains were counted from two flowers per sample.
The hypertriploid plant was crossed reciprocally with Clark 63. The hybridization procedure was the same as that reported by Singh et al. (1998). The young flower buds were emasculated and pollinated immediately with pollen from respective male plants. At maturity, both crossed and selfed pods from the hypertriploid plant and from the control plant (disomic sib) were harvested. Total number of pods and seeds from self-pollination were counted, and average number of seeds per pod was calculated.
Chromosome numbers of the F1 and selfed seeds harvested from the hypertriploid plant were examined according to the procedure described by Singh et al.(1998) and Xu et al. (1998). The seeds were germinated in a sand bench in the greenhouse. Root tips from actively growing 7- to 10-d-old seedlings were collected and pretreated with 0.05% (w/v) 8-hydroxyquinoline for 4 to 5 h at 16°C. Pretreated root tips were fixed in a 3:1 (v/v) mixture of 95% (v/v) ethanol and propionic acid for 24 h. Root-tips were hydrolyzed in 1 M HCl for 11 to 15 min at 60°C and stained in Schiff's reagent for 2 h at room temperature. Feulgen stain was removed and the root tips were rinsed with cold double-distilled water and stained with Carbol fuchsin stain overnight at 0 to 4°C in a refrigerator. After staining, the root tips were washed three to four times with cold double-distilled water and stored in cold double-distilled water in a refrigerator. Root tips were squashed in 45% (v/v) acetic acid.
Meiotic and mitotic observations were made with a Nikon Optiphot-2 Microscope (Nikon Inc., Instrument Group, Garden City, NY). Photomicrographs were taken under 100x oil plane objective using Kodak Technical Pan film 2415 (Eastman Kodak Co., Rochester, NY). The photomicrographs were enlarged and printed on Kodak Polycontrast III RC Glossy Paper (Eastman Kodak Co.).
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Results and discussion |
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TOPABSTRACTINTRODUCTIONMaterials and methodsResults and discussionREFERENCES |
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Our report is the first case of a hypertriploid (2n = 3x + 1 = 61) spontaneously occurring in a non-male-sterile genotype in soybean. The mechanism of formation of the hypertriploid might be quite different from that of the triploids derived from the ms1ms1 plants. Chen and Palmer (1985) suggested that spontaneous triploids from ms1ms1 plants could be formed from the fusion of a 2n egg fertilized by an n sperm. It has been reported that the ms1ms1 plants were characterized by formation of coenocytic microspores, resulting from the failure of cytokinesis after Telophase II (Albertsen and Palmer, 1979; Chen and Palmer, 1985; Chen et al., 1987). In addition, the megaspore mother cells (MMCs) in ms1ms1 plants were observed to undergo meiotic division without cytokinesis after Telophase II and thus produced multiple nuclei in megagametophytes (Cutter and Bingham, 1977; Kennell and Horner, 1985; Chen and Palmer, 1985; Zhang and Palmer, 1990). It is assumed that the restitution of some nuclei in the coenocytic microspores and megagametophytes might be responsible for the formation of 2n or polyploid gametes in the ms1ms1 plants. Zhang and Palmer (1990) noted that 2n egg cells in ms1ms1 plants could be produced from fusion between haploid gametes that came from meiotic division of the MMCs.
We believe that the hypertriploid plant in our study originated due to the formation of an unreduced gamete. Because the hypertriploid was derived from the cross of T31 x T190-47-3, it is difficult to determine which parental line provided the unreduced gamete. We observed that T31 plants were highly cleistogamous when they were growing under day lengths of 14 to 16 h in the greenhouse. Occasionally, some T31 plants would produce a few open flowers, but the flowers generally did not have enough pollen. In this specific physiological condition, one possibility was that the hypertriploid was formed from the fusion of a 2n egg (2n = 40) from T31 and an n + 1 pollen (n + 1 = 21) from primary trisomic line T190-47-3.
In soybean, the frequency of the hypertriploidy is extremely low. Among 10 F1 plants from the cross of T31 x T190-47-3, we found only one hypertriploid plant. The nine other plants had 2n = 40 chromosomes. All 146 F1 plants involving T31 crosses with 14 other primary trisomics had either 2n = 40 or 2n = 41 chromosomes, as expected. Previously, we found a plant with 2n = 62 chromosomes among 32 plants derived from a 2n = 42 chromosome plant; unfortunately, the plant with 2n = 62 died in a greenhouse accident. Additionally, we have not observed any triploids among about 3000 plants from the progenies of primary trisomics.
In the greenhouse, the hypertriploid plant was more vigorous in growth and matured much later than its disomic sib. The delayed maturity may be related to reduced seed set. The hypertriploid had robust main branches and large, dark-green, leathery leaves (Table 1) .
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The maximum chromosome configuration (1 IV + 19 III) was not observed in soybean hypertriploid, but it was reported in a number of the PMCs in barley hypertriploid (Singh and Tsuchiya, 1975). These differences are probably due to the difference in chromosome size between soybean and barley. The chromosomes in soybean are about 10 times smaller than those in barley. Presumably, the quadrivalent in soybean has a tendency to dissociate into two bivalents. Chromosome configurations in soybean tetrasomics (2n = 42) were mainly 21 II, less frequently 1 IV + 18 II or 1 III + 18 II + 1 I (S.J. Xu, 1999, unpublished results).
Chen and Palmer (1985) analyzed meiotic pairings in the autotriploids (2n = 3x = 60) derived from the genetic male-sterile (ms1ms1) soybean. They reported enneavalent (association of three trivalents), hexavalent, pentavalent, and quadrivalent configurations at diakinesis or Metaphase I. They proposed that these chromosome configurations were due to nonhomologous association, close secondary association, partial homology, and chromosome stickiness. However, except for the expected one quadrivalent, other multivalents were not observed in the studied hypertriploid. This discrepancy may be due to the different genetic backgrounds of the hypertriploid and the triploids derived from the ms1ms1 plants.
The soybean hypertriploid had a small number of the PMCs with a few more bivalents. Similar results were observed by Chen and Palmer (1985). These bivalents might be caused by either chromosome stickiness or possible nonhomologous pairing. Apparently, it is impossible to confirm the occurrence of nonhomologous chromosome associations in the soybean because some chromosomes were often sticking together at early Metaphase I plate. Thus, we analyzed the PMCs in the middle or late Metaphase I when the chromosomes showed the optimal spreading. Even in these cells, it was sometimes difficult to distinguish a bivalent from the two univalents that were sticking together because of the small chromosome size. Precocious separation in some bivalents and trivalents was common in some PMCs. This could be the reason that the PMCs with complete meiotic pairing were not observed.
The chromosome segregation at Anaphase I was analyzed only in 12 PMCs because it was difficult to find Anaphase I cells. Five cells showed 30 to 31 chromosome distribution (Fig. 1c), one showed 33 to 28, and the remaining six had one to four lagging chromosomes (Fig. 1d).
Pollen grains from the hypertriploid were easily classified into four distinct types: plump and fully stained, darkly stained, partially stained, and unstained (Table 1), similar to the pollen from the fertile Ms1ms1ms1 triploids reported by Chen and Palmer (1985). Sixty-three percent of pollen from the hypertriploid were fully stained as compared with 98% from the control. In addition, 15% of the pollen grains from the hypertriploid were darkly stained, whereas this type of pollen was not observed in the control (Table 1). The first flush of flowers and some late flowers on the hypertriploid generally aborted. However, the hypertriploid plant produced a total of 98 selfed seeds (Table 1). Sixteen seeds were produced from 147 cross-pollinations of hypertriploid (
) x Clark 63 (
); however, seed set was not obtained from 56 cross-pollinations of the reciprocal cross.
The soybean hypertriploid in our study showed a similar frequency of pollen fertility (63%) to the male-fertile autotriploid (Ms1ms1ms1) plants (57–82%) reported by Chen and Palmer (1985). However, the hypertriploid set a much higher number of seeds (98) than the Ms1ms1ms1 autotriploid plants where seed set ranged from 1 to 13, with an average of 4.4 seeds per plant (Chen and Palmer, 1985). This may be attributed to the different genetic backgrounds of the hypertriploid and Ms1ms1ms1 autotriploid.
The autotriploids generally had poor seed set from self-pollination even though they may have high pollen fertility such as in barley (Tsuchiya, 1952), jimson weed (Datura stramonium L.) (Satina and Blakeslee, 1937), einkorn (Triticum monococcum L.) (Kuspira et al., 1986), and pearl millet [Pennisetum glaucum (L.) R. Br.] (Dujardin and Hanna, 1988). It has been suggested that the low seed fertility of the autotriploids can be attributed to the imbalance of gamete chromosome number, or failure of zygotic and endosperm development (Schulz-Schaeffer, 1980; Chen and Palmer, 1985). The high seed set of the soybean hypertriploid may be related to certain meiotic factor(s). The meiotic pairing analysis in our study showed that the hypertriploid had a relatively high frequency of bivalents. The high number of bivalents should help in producing more chromosomally balanced gametes.
Chromosome constitution was determined in the progenies of the hypertriploid (Table 3) . Twelve of 16 crossed (F1) seeds germinated and all were pubescent. The chromosome numbers of the plants ranged from 2n = 40 to 2n = 48, with the exception of one plant with 2n = 56 (Figs. 2a, 2b, 2c) . The seeds from self-pollination of the hypertriploid germinated poorly and only 43 (44.0%) of 98 seeds could germinate. The regular plump seeds germinated but most of the shrunken and irregular-shaped seeds were not viable, suggesting that the low germination rate of the seeds was caused by the poor development of endosperm. The chromosome numbers of the plants from self-pollination ranged from 2n = 50 to 2n = 69 (Fig. 2d). This result was similar to that of the selfed progenies from Ms1ms1ms1 autotriploids (Chen and Palmer, 1985). The difference was that the plants with 2n = 40 to 2n = 49 were not found in selfed progenies of the hypertriploid. The segregation of glabrous trait in selfed population was not recorded because plants were weak and died at seedling stage.
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) x 2x () and the reciprocal cross in spinach ranged from diploid (2n = 2x = 12) to triploid (2n = 3x = 18) (Janick et al., 1959). We observed high seed fertility in some plants with 2n = 44 to 2n = 47 isolated from the hypertriploid x diploid in soybean. Therefore, the high seed fertility in soybean hypertriploid and its progenies suggests that soybean male and female spores may tolerate a higher number of extra chromosomes than most other diploid species. The tolerance of extra chromosomes by soybean male and female spores was also supported by the high levels of male and female transmission of the extra chromosome in primary trisomics (2n = 41) of soybean. Palmer (1976) reported 34 to 45% ovule transmission and 22 to 43% pollen transmission in three primary trisomics. However, primary trisomics hardly transmit their extra chromosomes through pollen in a majority of other diploid species. The tolerance is probably because soybean contains a higher number of chromosomes (2n = 40) compared with other diploid species such as barley (2n = 14), rice (Oryza sativa L., 2n = 24), tomato (Lycopersicon esculentum Mill., 2n = 24), and maize (Zea mays L., 2n = 20). Also, the high seed fertility of hypertriploid and the tolerance of more extra chromosomes might be related to the polyploid nature of soybean. It has been proposed that soybean is a diploidized tetraploid, which cytogenetically acts like a diploid (Hadley and Hymowitz, 1973; Palmer, 1976; Crane et al., 1982; Lee and Verma, 1984). However, the hypothesis has not been proved by direct evidence (Chen and Palmer, 1985). Although soybean is one of several economically important crops, its cytogenetics has lagged far behind that of other major crops (Palmer and Kilen, 1987). The hypertriploid is unique material for broadening our understanding of the cytological and genetic behavior of the soybean.
The plants from self-pollinated hypertriploid contained a high number of chromosomes and most of them could not grow to maturity, probably because of unbalanced chromosome constitutions. Therefore, they are not applicable to produce primary trisomics (2n = 41) in soybean. In contrast, the F1 plants derived from hypertriploid x diploid had a relatively low number of chromosomes. Some of these plants had high seed fertility and their progenies would segregate for plants with different chromosome numbers. Therefore, a large number of primary trisomic lines (2n = 41) could be isolated from the progenies of these plants by consecutive self-pollination or by hybridization with disomic plants. They complement the aneuploids derived from male-sterile lines in developing a complete set of primary trisomics in soybean.
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ACKNOWLEDGMENTS |
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Received for publication April 19, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMaterials and methodsResults and discussionREFERENCES |
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