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Published in Crop Sci. 43:1741-1746 (2003).
© 2003 Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA

CROP BREEDING, GENETICS & CYTOLOGY

Morphological Comparison of Progeny Derived from 4x-2x and 4x-4x Hybridizations of Lotus glaber Mill. and L. corniculatus L.

P. R. Beuselinck*,a, J. J. Steinerb and Y. W. Rimc

a USDA-ARS, Plant Genetics Res. Unit, Univ. of Missouri, Columbia, MO 65211
b USDA-ARS, National Forage Seed Production Res. Center, Corvallis, OR 97331
c Livestock Res. Inst., Grass and Forage Division, KyunSunGu Suwon, Korea 441-350

* Corresponding author(beuselinckp{at}missouri.edu).


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 NOTES
 RESULTS AND DISCUSSION
 REFERENCES
 
Unreduced gametes (2n gametes) from Lotus glaber Mill. (2n = 2x = 12) may aid intercrossing with L. corniculatus L. (2n = 4x = 24) to produce progeny with a greater heterotic advantage than progeny derived from colchicine-induced {7-acetamido-6,7-dihydro-1,2,3,10-tetramethoxybenzo[a]heptalen-9(5H)-one} tetraploid (4x) L. glaber. The objective of this research was to compare progeny produced from 4x with 2x (4x-2x) and 4x with 4x (4x-4x) crosses and determine if 2n gametes had a heterotic advantage over normal gametes derived from colchicine-induced tetraploids. Vegetative cuttings of the 2n-gamete-producing L. glaber genotype 204882-1 (2x 204882-1) were treated with colchicine and successfully yielded an autotetraploid (2n = 4x = 24) of 204882-1 (4x 204882-1). Three randomly chosen genotypes from the L. corniculatus germplasm MU-81 were used as maternal parents in crosses to 2x 204882-1 and 4x 204882-1. Progeny produced from 4x-2x crosses were intermediate to or larger than their parents in their leaf and flower morphology. Progeny from 4x-4x crosses were intermediate to or smaller than their parents in leaf and flower size. Size of leaves and flowers of progeny from 4x-2x crosses were similar to or larger than 4x-4x progeny. Morphological comparisons demonstrated that heterotic expression of progeny from 4x-2x crosses was greater than progeny from 4x-4x crosses. The results indicate that the generation of interspecific L. corniculatus x L. glaber hybrids via 2n pollen to be effective, but a means of exploiting any heterotic advantage in these cross-pollinated species needs to be identified.

Abbreviations: RAPD, random amplified polymorphic DNA


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 NOTES
 RESULTS AND DISCUSSION
 REFERENCES
 
LOTUS CORNICULATUS is a tetraploid (2n = 4x = 24), cross-pollinated perennial herbage legume grown for hay and pasture. The genetic improvement of cultivated L. corniculatus in the USA has been limited by the lack of genetic variability (Beuselinck et al., 1984) and the continued cultivar selection from a narrow germplasm base (Steiner and Poklemba, 1994). Breeders have tried to overcome specific trait limitations through interspecific hybridization, including attempts to reduce seed shattering by transferring pod indehiscence from L. conimbricensis Brot. and L. ornithopodiodes L. (O'Donoughue and Grant, 1988); improved seedling vigor from L. glaber (Bent, 1962); active winter growth through reduced winterhardiness (Negri and Veronesi, 1986); and rhizomes from L. uliginosus Schkur. (Gershon, 1961). However, these attempts to introduce desirable traits into L. corniculatus from diploid Lotus species have yielded little success.

Two methods of interest have been described to double the chromosome number of 2x diploid Lotus species (2n = 2x = 12 or 14) for germplasm transfer to L. corniculatus: (i) autotetraploid production by colchicine-induced chromosome doubling of 2x species, and (ii) 2n gametes to create 4x interspecific hybrids between 2x and 4x species. Polyploidization can be achieved by somatic doubling of the chromosomes (asexual polyploidization) or functional production of 2n gametes by aberrations in the meiotic process by sexual polyploidization (Camadro and Peloquin, 1980). Although both modes can double chromosome number, their relative efficacy becomes evident when genetic variability, inbreeding, heterozygosity, and epistasis are considered (Camadro and Peloquin, 1980; Iwanaga and Peloquin, 1982). Almost all polyploid angiosperms arose primarily through sexual reproduction from 2n gametes rather than through spontaneous doubling of somatic chromosomes (Harlan and de Wet, 1975).

Amphidiploid development from the interspecific hybridization of 2x Lotus species followed by colchicine-induced autotetraploidy has been successful (Somaroo and Grant, 1971; O'Donoughue and Grant, 1988), and resulting amphidiploids have been crossed with L. corniculatus (Somaroo and Grant, 1972). Limited germplasm transfer between 2x and 4x species should be enhanced with colchicine; however, few successful hybrids have been produced (Phillips and Keim, 1968; de Lautour et al., 1978; Grant, 1999).

Sexual polyploidization through 2n gametes has been a major route for naturally occurring polyploids (Harlan and de Wet, 1975). Unreduced gametes can be used to overcome otherwise infertile interploidy crosses and efficiently transfer germplasm from wild relatives to cultivated species, especially from 2x and 4x species (Havey and Maxwell, 1988). Production of 2n gametes has been extensively studied in potato (Solanum tuberosum L.; Mok and Peloquin, 1975b; Camadro and Peloquin, 1980), alfalfa (Medicago sativa L.; McCoy and Rowe, 1986; Veronesi et al., 1986), red clover (Trifolium pratense L.; Parrott and Smith, 1984), and pea (Pisum sativum L.; Myers et al., 1984). Unreduced gametes are considered to be more desirable for crossing with 4x species than gametes produced from colchicine-induced tetraploids if a heterotic advantage can be exploited.

Negri and Veronesi (1986) reported the production of 2n pollen by 2x L. glaber based on the occurrence of 4x progeny from 4x with 2x crosses between L. corniculatus and L. glaber, but no further progress has been reported. Rim and Beuselinck (1996) identified three different 2n gamete forms in L. glaber, and determined that the genotype 204882-1 produced up to 6% 2n pollen. Diploid Lotus spp. capable of producing 2n gametes could facilitate interspecific 4x-2x crossing with L. corniculatus and further L. corniculatus germplasm improvement. The objective of this research was to compare progeny produced from 4x by 2x (4x-2x) and 4x by 4x (4x-4x) crosses, and determine if 2n gametes had a heterotic advantage over normal gametes derived from colchicine-induced tetraploids in the hybrid generation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
NOTES
 RESULTS AND DISCUSSION
 REFERENCES
 
Lotus-glaber-Induced Autotetraploidy
Diploid L. glaber genotype 204882-1 (2x 204882-1) is described elsewhere (Rim and Beuselinck, 1996). Vegetative stem cuttings of 2x 204882-1 were rooted in vermiculite at 24°C and 24-h daylength in a growth chamber providing 350 µmol photon m-2s-1 of fluorescent–incandescent lighting. After roots developed, apical meristems of the cuttings were submerged in one of six colchicine solutions (0.1, 0.2, 0.3, 0.4, 0.5, and 1.0 mg L-1) for six exposure-period treatments (3, 5, 6, 7, 10, or 20 h). Colchicine-treated cuttings were washed with tap water for 3 to 4 min, then transplanted into a commercial soil mix (Promix, Premier Horticulture, Dorval, QC, Canada)1, and grown in a greenhouse. Cuttings were examined after four months for mitotic chromosome numbers (Rim and Beuselinck, 1996). Tentative autotetraploids were verified by meiotic examination with anthers excised from young flowers. Chromosome pairing configurations were determined from 10 well-spread cells at diakinesis for the number of univalents, bivalents, trivalents, and quadrivalents per cell.

Verified, induced 4x clones were morphologically compared with 2x 204882-1 clones and three randomly chosen genotypes of L. corniculatus cv. MU-81 (Beuselinck and McGraw, 1986). Lengths and widths of central leaflets from the first to third node from the stem apex were measured on 10 stems collected at random from each plant. Ten umbels were collected at random from each plant, and floral bract and standard lengths and widths; and calyx, calyx tube, style, and ovary lengths were measured on one flower from each umbel. Indices were calculated for: (i) central leaflet (width/length of central leaflet), (ii) floral bract (width/length of floral bract); (iii) standard (width/length of standard); (iv) calyx (calyx tube length/total calyx length); and (v) style/ovary (length of style/length of ovary).

Pollen morphology of 2x 204882-1, 4x 204882-1, and MU-81 was compared with light-transmission microscopy (Rim and Beuselinck, 1996). Length and width of 50 fresh pollen grains from each genotype were measured with an ocular micrometer. Analyses of variance were performed on length and width of pollen, and means were compared with Fisher's protected LSD test when the F test showed significance at P <= 0.05.

Pollen germination from 4x 204882-1 and 2x 204882-1 was compared for each plant with fresh pollen collected from one floret of each of five umbels. Pollen samples were mixed together, distributed in a few drops of B5 medium (Gamborg et al., 1968), spread on a glass slide, incubated at 25°C, and germination examined 3 to 6 h later. Viable pollen grains were evidenced by the presence of a pollen tube and expressed as a percentage of pollen that germinated.

Lotus corniculatus x L. glaber Crosses
Flowers of the MU-81 were emasculated by hand 1 to 2 d before anthesis, and then hand-pollinated 1 to 2 d later with pollen of 2x 204882-1 or 4x 204882-1. Pods were harvested at maturity and the number of pods produced per pollinated flower and number of seeds per pod were recorded. Seeds obtained from crosses were mechanically scarified and then germinated on B5 culture medium (Gamborg et al., 1968). Germinated seedlings were transferred to a commercial soil mix and grown in a greenhouse.

Hybridization in progeny was verified by random amplified polymorphic DNA (RAPD) banding patterns (Nualsri et al., 1998). DNA was isolated from leaf tissues of each parent and putative hybrid progeny with an extraction buffer containing hexadecyltrimethyl-ammonium bromide (CTAB) (Doyle and Doyle, 1990). DNA extraction from tissues and RAPD analysis were the same as described by Beuselinck et al. (1996). Four 10-base oligonucleotide primers (OPB-17, OPB-20, OPC-19, and OPF-18, Operon Technologies, Alameda, CA) were used for discriminating among hybrids. Hybridization was assumed to have occurred when at least one band unique to each parent was found in a progeny. Hybrid pollen viability was determined as described above. Female progeny fertility was determined by pollinating 20 emasculated flowers with mixed pollen from all MU-81 clones.

Morphological traits were measured on the 4x-4x and 4x-2x progeny as previously described for parents. Analyses of variance were performed on means of morphological data for parents and their 4x-4x and 4x-2x progenies. A general linear model (SAS Institute, 1988) analysis was performed on morphological data means and midparent vs. their F1 progeny. Mean differences were determined with Fisher's protected least significant difference test. All differences are significant at P <= 0.05 unless otherwise indicated.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 NOTES
 RESULTS AND DISCUSSION
REFERENCES
 
Six clones from 2x 204882-1 vegetative stem cuttings exhibited a somatic chromosome number of 2n = 24 as a result of the colchicine treatments. Meiotic examination revealed that only one of these clones, treated with 1.0 mg L-1 colchicine for 20 h, acted as a true tetraploid; the others acted as diploids or mixoploids. The tetraploid clone was designated 4x 204882-1.

2x 204882-1 and 4x 204882-1
The 24 somatic chromosomes of MU-81 usually paired as 12 bivalents, although an occasional quadrivalent was observed (Table 1). These results agree with those of Wernsman et al. (1964)(1965). The 12 somatic chromosomes of 2x 204882-1 paired as six bivalents without exception. Colchicine-induced 4x 204882-1 produced an equal number of rod and ring bivalents and few quadrivalents. No univalents or trivalents were observed in 4x 204882-1. Somaroo and Grant (1971) reported 4x L. glaber showed a mean frequency of 8.97 bivalents (range 2 to 12) and 1.0 quadrivalent (range 0 to 5), where a theoretical maximum of 6.0 quadrivalents would be expected. This result is similar to that observed in the meiotic chromosome pairing of 4x 204882-1.


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  		Table 1. Chromosome behavior at diakinesis of MU-81 Lotus corniculatus, diploid L. glaber accession 204882-1 (2x 204882-1), autotetraploid L. glaber 204882-1 (4x 204882-1), six progeny from MU-81 x 2x 204882-1, and 15 progeny from MU-81 x 4x 204882-1.

 
Morphological changes are typical of induced polyploidy that results from increased chromosome numbers (Drobet and Pestova, 1980; Ramachandran, 1982). The 2x 204882-1 and 4x 204882-1 clones differed for all measured morphological characters except the central leaflet and style/ovary indices (Table 2). Differences in leaf characters between 2x and 4x 204882-1 were also significant, while differences in central leaflet indices were not. The mean length and width of the central leaflet of 4x 204882-1 were {approx}1.4- and 1.2-fold greater than leaves of 2x 204882-1, respectively. Size of the floral parts of 4x 204882-1 were {approx}1.2- to 1.7-fold larger than 2x 204882-1. Lengths of central leaflets, floral bracts, standards, calyx tubes, and styles of 4x 204882-1 were not different from those of MU-81.


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  		Table 2. Means for somatic chromosome number and pollen germination, and presence of 2n pollen and pod production for three genotypes of MU-81 Lotus corniculatus, diploid L. glaber accession (2x 204882-1), autotetraploid L. glaber 204882-1 (4x 204882-1), six progeny from MU-81 x 2x 204882-1, and 16 progeny from MU-81 x 4x 204882-1.

 
Pollen of 4x 204882-1 was morphologically similar but larger than pollen of 2x 204882-1. Length and width of pollen averaged 1.56 and 1.58 times greater than 2x 204882-1 pollen, respectively. Mean pollen length and width of 4x 204882-1 were 21.9µ (SE ± 0.12µ) and 17.3µ (SE ± 0.09µ), respectively, while mean length and width of 2x 204882-1 pollen were 14.0µ (SE ± 0.11µ) and 11.0µ (SE ± 0.09µ). Germination percentage of 2x 204882-1 pollen was 90%, while germination of 4x 204882-1 pollen was 32%.

Lotus corniculatus x L. glaber Crosses
Crosses between L. corniculatus MU-81 and L. glaber 2x 204882-1 yielded 17 seeds from 692 pollinated MU-81 flowers, and the 17 seeds produced seven seedlings. Low seed set (mean 0 to 1.5 seeds per flower pollinated) among 4x-2x crosses likely resulted from a low frequency of 2n pollen produced by 2x 204882-1. Rim and Beuselinck (1996) reported the frequency of 2n pollen to be <=6%. A positive relationship between 2n pollen frequency and seed set in 4x-2x crosses has been demonstrated in alfalfa (McCoy, 1982).

Six of the seven 4x-2x progeny were tetraploid and one was triploid (2n = 18) (Table 3). The low number of triploids suggests L. corniculatus has an effective triploid block. Triploid blocks in interploid crosses have been found in several other species and result from abortion of triploid embryos due to endosperm imbalance (Johnston et al., 1986). All seven 4x-2x progeny expressed RAPD bands unique only to 2x 204882-1 used as the paternal parent.


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  		Table 3. Comparisons of morphological characters for MU-81 Lotus corniculatus, diploid L. glaber accession 204882-1 (2x 204882-1), autotetraploid L. glaber 204882-1 (4x 204882-1), 4x-2x progeny from MU-81 x 2x 204882-1, 4x-4x progeny from MU-81 x 4x 204882-1, and midparent vs. F1.

 
Pollen germination percentage in the seven 4x-2x progeny ranged between 0 to 85% (Table 3), while the parent values averaged 55% for MU-81 and 90% for 2x 204882-1, respectively. The one triploid progeny produced non-viable pollen. Fertility of the other six 4x-2x progeny varied when pollen was tested, at times being either greater or less than the parental germination percentages.

The six progeny from the 4x-2x crosses had 24 somatic chromosomes that behaved like those of MU-81, although they exhibited greater univalent and quadrivalent frequencies (Table 1). The chromosome behavior expressed by the progeny in this study was similar to observations on L. corniculatus x 4x L. glaber hybrids made by Wernsman et al. (1965) where the 24 somatic chromosomes usually paired as 12 bivalents, although occasional quadrivalents were found. Formation of univalents in three of the six 4x-2x progeny indicated nonhomology between chromosomes of the L. corniculatus and L. glaber genomes. This evidence suggests L. corniculatus did not originate by L. glaber autotetraploidy, which is supported by the findings of Steiner (1999). The meiotic configurations suggest that bivalent chromosome pairing predominates. The occurrence of about half rod bivalents is evidence that there may be insufficient chiasmata to hold quadrivalents together, as is the case in alfalfa (McCoy and Bingham, 1988). A high number of bivalents is typical in L. corniculatus, but Fjellstrom et al. (2001) provided RFLP evidence of tetrasomic inheritance to support an autotetraploid classification of L. corniculatus. The tetrasomic genetic ratios provide further evidence that the bivalent pairing in Lotus is random, similar to alfalfa and potato (McCoy and Bingham, 1988; Mendiburu and Peloquin, 1977).

It did not appear that there was a relationship between meiotic configurations and pollen germination among the 4x-2x progeny. Some progeny exhibited a low pollen germination percentage without observed meiotic irregularity, as well as the opposite case, where pollen from progeny with high meiotic irregularities germinated well (Table 2).

The 4x-2x progeny leaf and flower characters differed from their parents (Table 3). Lengths and widths of leaves and flowers of MU-81, except width of floral bracts, differed from that of 2x 204882-1, and 4x-2x progeny morphology was generally intermediate to the parents. Midparent values for all leaf and floral characters, except the indices (floral bract, calyx, and pistil), differed from values of the 4x-2x progeny. The results indicate tetraploid progeny from crosses between MU-81 and 2x 204882-1 showed a heterotic response over their parents in leaf and flower size.

Good seed set was obtained from the cross MU-81 x 4x 204882-1. The great number of seed (223) obtained from the pollination of 37 MU-81 flowers with pollen from 4x 204882-1 indicated considerable homology between the MU-81 and 4x 204882-1 genomes. Sixteen progeny were obtained from 20 randomly chosen seeds and the seedlings did not require special culture conditions. All 16 progeny were verified as hybrids by RAPD markers with five primers expressing bands unique only to 4x 204882-1.

Pollen germination from the 4x-4x progeny varied between nonviable and high germination (Table 2). Pollen viability from interspecific 4x-4x Lotus progeny has been reported to be lower than their parents (O'Donoughue and Grant, 1988). However, in this random set of 4x-4x progeny, pollen viability for some progenies was greater than that of their parents. All but two of the 16 MU-81 x 4x 204882-1 progeny produced pods when pollinated with MU-81 pollen, with one progeny completely sterile and the other failing to produce any viable pollen or pods.

Progeny from 4x-4x crosses differed for leaf and flower characters compared with their parents (Table 3). Progeny central leaflet length, floral bract and standard size, and length of calyx and pistil were generally smaller than either parent. The 4x-4x progeny morphology did not exceed either parent except for leaf width. All leaf and floral character values of the 4x-4x progeny, except central leaflet width, were smaller (P <= 0.01) than the midparents' values for each character. These results are dissimilar to reports by Bent (1962) and Wernsman et al. (1965) who showed interspecific L. corniculatus x 4x L. glaber progeny were intermediate to the parental species.

4x-2x and 4x-4x Progeny Comparisons
All leaf and floral characters except central leaflet width and total calyx length were larger for the progeny from MU-81 x 2x 204882-1 than from MU-81 x 4x 204882-1 (Table 3). Size differences between 4x-2x and 4x-4x progeny could be an expression of an advantage of unreduced (2n) gametes from 204882-1 over haploid gametes from 4x 204882-1. Mean midparent values for leaf and floral characters compared with 4x-2x progeny values were significantly different (P <= 0.001), indicating a heterotic advantage for 2n gametes. Unreduced gametes are considered to be more desirable than doubled gametes from colchicine-induced polyploids when maximizing heterozygosity (Mok and Peloquin, 1975a; McCoy and Rowe, 1986). However, unreduced gametes are not advantageous unless they are generated by superior genotypes.

With the exception of one hybrid, no 4x-2x hybrid exceeded the morphological performance of both parents. It appears that any genetic advantage from a 2x 204882-1 2n gamete was not large enough to compensate for the genotypic inferiority of this diploid L. glaber parent relative to the L. corniculatus parent. Although morphological comparisons demonstrated that heterotic expression was greater for 4x-2x progeny than 4x-4x progeny, neither progeny as a group exceeded the L. corniculatus parent. The results indicate interspecific L. corniculatus x L. glaber hybrids via 2n pollen was effective and demonstrated an advantage over hybrids produced via colchicine-induced 4x L. glaber. However, low seed number production from 4x-2x crosses due to low 2n pollen production, the difficulty in locating superior 2n gamete producing genotypes, and the reproductive behavior of L. corniculatus may limit the utility of this approach. The relative ease of developing colchicine-induced autotetraploids of L. glaber and the high success rate from 4x-4x crosses counter arguments for using 2n gametes from L. glaber. Any heterotic potential is best exploited in a hybrid, especially in a species that is able to propagate via vegetative reproduction, like potato. Lotus corniculatus can produce rhizomes (Beuselinck et al., 1996), a form of vegetative reproduction. However, the rhizomes cannot be used to exploit a heterotic advantage that could be derived via 2n gametes from L. glaber because L. corniculatus is disseminated by seed and cross-pollination after the initial hybrid generation would eliminate heterosis.

The feasibility of improving interspecific germplasm transfer by sexual polyploidization with 2n gametes or induced autoploidy is presented in this study. More extensive study of diploid Lotus spp. producing 2n gametes that could facilitate interspecific 4x-2x, 2x-4x crossing with L. corniculatus is needed. Before significant contributions could be made to L. corniculatus germplasm improvement via 2n gamete producing diploid Lotus species, a means of exploiting any heterotic advantage needs to be identified.


   NOTES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 NOTES
RESULTS AND DISCUSSION
 REFERENCES
 
1 Mention of a trademark, vendor, or proprietary product does not constitute a guarantee or warranty of the product by the USDA or the Univ. of Missouri and does not imply its approval to the exclusion of other products or vendors that may also be suitable. Back

Received for publication October 9, 2002.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 NOTES
 RESULTS AND DISCUSSION
 REFERENCES
 





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