ORIGIN OF FRAGARIA POLYPLOIDS. II. UNREDUCED AND DOUBLED-UNREDUCED GAMETES

Offspring from natural hybrids between octoploid Fragaria chiloensis (2n = 56) and diploid F. vesca (2n = 14) backcrossed under natural conditions to F. chiloensis were studied. The natural F₁ hybrids themselves were of three kinds: (1) The expected pentaploids which resulted from the union of normally reduced gametes of diploid F. vesca and octoploid F. chiloensis; (2) A hexaploid F₁ hybrid which resulted from the union of an unreduced gamete from diploid F. vesca with a normally reduced gamete from octoploid F. chiloensis; and (3) A 9-ploid F₁ hybrid which probably arose from the union of an unreduced gamete of the octoploid F. chiloensis with a normally reduced gamete of diploid F. vesca. The progenies that resulted from the natural backcrossing of each of the three sorts of F₁ hybrids to F. chiloensis were as follows: The pentaploid F₁ hybrids (2n = 35) yielded mostly 9-ploid offspring from unreduced 5X gametes; a relatively high percentage of 14-ploid plants arising from doubled-unreduced 10 X gametes and a few 2N = ±46 aneuploids from reduced gametes. The hexaploid F₁ hybrid (2n = 42) on backcrossing yielded over 50% 10-ploid offspring with the rest 2n = ±50 aneuploids from reduced gametes. The 9-ploid F₁ hybrid (2n = 63) on backcrossing yielded mostly aneuploids normally distributed about a modal 2n = 59 chromosome class resulting from a 31 chromosome gamete, with a few 2n = 56 and 2n = 63 euploids. The 9-ploids may facilitate diploid Å octoploid introgression. Screening of the open-pollinated offspring from F. chiloensis revealed almost 2% 12-ploid (2n = 84) offspring from the union of the reduced and unreduced F. chiloensis gametes. The probable genomic constitution of the observed novel ploidy levels and those that theoretically may be generated from the known hybrids are presented. The origin of the existing polyploids from diploids through simple unreduction is postulated.     

Chromosome elimination and fragment introgression and recombination producing intertribal partial hybrids from <Emphasis Type="Italic">Brassica napus</Emphasis> × <Emphasis Type="Italic">Lesquerella fendleri</Emphasis> crosses

The intertribal sexual hybrids between three Brassica napus (2n = 38) cultivars and Lesquerella fendleri (2n = 12) with the latter as pollen parent were obtained and characterized for their phenotypes and chromosomal and genomic constitutions. F₁ plants and their progenies mainly resembled female B. napus parents, while certain characters of L. fendleri were expressed in some plants, such as longer flowering period, basal clustering stems and particularly the glutinous layer on seed coats related to drought tolerance. Twenty-seven F₁ plants were cytologically classified into five types: type I (16 plants) had 2n = 38, type II (2) had 2n = 38–42, type III (3) had 2n = 31–38, type IV (5) had 2n = 25–31, and type V (1) had 2n = 19–22. Some hybrids and their progenies were mixoploids in nature with only 1–2 chromosomes or some chromosomal fragments of L. fendleri included in their cells. AFLP (Amplified fragments length polymorphism) analysis revealed that bands absent in B. napus, novel for two parents and specific for L. fendleri appeared in all F₁ plants and their progenies. Some progenies had the modified fatty acid profiles with higher levels of linoleic, linolenic, eicosanoic and erucic acids than those of B. napus parents. The occurrence of these partial hybrids with phenotypes, genomic and fatty acid alterations resulted possibly from the chromosome elimination and doubling accompanied by the introgression of alien DNA segments and genomic reorganization. The progenies with some useful traits from L. fendleri should be new and valuable resource for rapeseed breeding.     

Development of a ploidy series from a single interspecific Trifolium repens L.Ā . nigrescens Viv. F1 hybrid

 The objective of the current research was to generate a ploidy series of backcross progenies from a single triploid (2n=3x=24) Trifolium repens×T. nigrescens F₁ hybrid (3x H-6909-5). The 3x H-6909-5 plant was highly sterile and produced no seeds from approximately 3000 reciprocal backcrosses to both parental species. Chromosome doubling by an in vitro colchicine method resulted in a marked increase in fertility. Pollen stainability was increased from 9.9% in 3x H-6909-5 to an average of 89.2% (range 87.7–90.9%) in the three chromosome-doubled 6x H-6909-5 plants. Subsequent backcrosses of 6x H-6909-5 and interbreeding of backcross derivatives resulted in an array of fertile hybrids at 4x, 5x and 7x levels and some aneuploids. The occurrence of 7x BC₁F₁ progeny from the T. repens×6x H-6909-5 (4x×6x) cross is the first unequivocal evidence of functional female 2n gametes in white clover. Meiotic pairing in F₁ and BC₁F₁ progeny indicated the presence of allosyndetic pairing, suggesting that genetic exchange between the two species is possible. Received: 17 October 1996 / Accepted: 8 November 1996     

Development of monosomic alien addition lines and introgression of genes from Oryza australiensis Domin. to cultivated rice O. sativa L.

Oryza australiensis, a diploid wild relative of cultivated rice, is an important source of resistance to brown planthopper (BPH) and bacterial blight (BB). Interspecific hybrids between three breeding lines of O. sativa (2n=24, AA) and four accessions of O. australiensis (2n=24, EE) were obtained through embryo rescue. The crossability ranged from 0.25% to 0.90%. The mean frequency of bivalents at diakinesis/metaphase I in F₁ hybrids (AE) was 2.29 to 4.85 with a range of 0–8 bivalents. F₁ hybrids were completely male sterile. We did not obtain any BC₁ progenies even after pollinating 20,234 spikelets of AE hybrids with O. sativa pollen. We crossed the artificially induced autotetraploid of an elite breeding line (IR31917-45-3-2) with O. australiensis (Acc. 100882) and, following embryo rescue, produced six F₁ hybrid plants (AAE). These triploid hybrids were backcrossed to O. sativa. The chromosome number of 16 BC₁ plants varied from 28 to 31, and all were male sterile. BC₂ plants had 24–28 chromosomes. Eight monosomic alien addition lines (MAALs) having a 2n chromosome complement of O. sativa and one chromosome of O. australiensis were selected from the BC₂ F₂ progenies. The MAALs resembled the primary trisomies of O. sativa in morphology, and on the basis of this morphological similarity the MAALs were designated as MAAL-1, -4, -5, -7, -9, -10, -11, and -12. The identity of the alien chromosome was verified at the pachytene stage of meiosis. The alien chromosomes paired with the homoeologous pairs to form trivalents at a frequency of 13.2% to 24.0% at diakinesis and 7.5% to 18.5% at metaphase I. The female transmission rates of alien chromosomes varied from 4.2% to 37.2%, whereas three of the eight MAALs transmitted the alien chromosome through the male gametes. BC₂ progenies consisting of disomic and aneuploid plants were examined for the presence of O. australiensis traits. Alien introgression was detected for morphological traits, such as long awns, earliness, and Amp-3 and Est-2 allozymes. Of the 600 BC₂ F₄ progenies 4 were resistant to BPH and 1 to race 6 of BB. F₃ segregation data suggest that earliness is a recessive trait and that BPH resistance is monogenic recessive in two of the four lines but controlled by a dominant gene in the other two lines.     

Genetic studies of self-fertility in rye (Secale cereale L.). 1. The identification of genotypes of self-fertile lines for the Sf alleles of self-incompatibility genes

Segregation for self-fertility has been studied in progenies from the crosses of self-sterile (SS) plants with interline hybrids obtained by a diallel scheme of pollinations between seven self-fertile (SF) lines (nos. 2–8) and with F₁ (SS plant x SF line) hybrids. All the offspring families from the SS plant x F₁ (SS plant x SF line) crosses demonstrated a 1SF1SS segregation. The crosses of SS plants with some interline hybrids gave only self-fertile plants, whereas the crosses with other interline hybrids gave a segregation of 3SF:1SS expected in the case of digenic segregation. The data obtained permitted us to identify three different S loci (S1, S2, S5) and to estimate the genotypes of self-fertile lines for their Sf alleles: lines 5, 6, 7 and 8 are S1f/S1f S2n/S2n S5m/S5m, line 4 is S1n/S1n S2f/S2f S5m/S5m, and lines 2 and 3 are S1n/S1n S2m/S2m S5f/S5f(Sn, Sm designate active alleles of the incompatibility genes). The identification of the particular S gene which is presented by the Sf allele in each line has been made on the basis of our data concerning the linkage of the Sf mutation with isozyme markers of particular rye chromosomes, which is reported in an accompanying paper.     

ARTIFICIAL HYBRIDIZATION OF RUBBER-BEARING GUAYULE WITH COLD-TOLERANT PARTHENIUM LIGULATUM

ABSTRACT Caespitose and cold-tolerant plants of Parthenium ligulatum (Jones) Barneby (Asteraceae) from a native population in the Uinta Basin, Utah, were uprooted, potted, and transferred to a greenhouse in California. Approximately two years after transfer, the plants flowered and subsequently were crossed to diploid guayule (Parthenium argentatum Gray), the rubber-bearing species, native to the state of Durango, Mexico. Only female guayule × male P. ligulatum crosses produced F₁ hybrids. Only crosses involving guayule as female parent and F₁ plants as male parents produced backcross (BC,) plants. Hybrid plants were variable with respect to their growth habit, inflorescence, and leaf shape. Both parents and F₁ hybrids had 2n = 36 chromosomes. Unlike the parents, however, meiosis was irregular in the hybrids which showed a range of 0–5 and an average of 2.1 univalents at metaphase I. Hybrids averaged 0.87 laggards at anaphase I and 0.83 micronuclei at the tetrad stage. The crossability of guayule and P. ligulatum, the high degree of chromosome pairing of the F₁ hybrids, and the production of BC₁ plants indicate that the two species are related in spite of their distinct morphological and ecological differences. This study suggests that the cold-tolerance trait of P. ligulatum may be transferred to guayule through interspecific hybridization followed by backcrossing. The development of cold-tolerant guayule cultivars is expected to expand the areas of guayule production beyond that of the Chihuahuan desert and similar climates.     

Interspecific hybrids between Brassica napus L. and B. oleracea L. developed by embryo culture

Summary Interspecific hybrids between Brassica napus and B. oleracea are difficult to produce, and previous attempts to transfer economic characters from one species to the other have largely been unsuccessful. In these studies, oilseed rape cv. Tower (2n38) (B. napus) was crossed with broccoli and kale (2n18) (B. oleracea), and hybrid plants were developed from embryos in culture by either organogenesis or somatic embryogenesis. In rape × broccoli, F₁ plants were regenerated from hybrid embryos and the plants produced viable selfed seeds. F₅ plants (2n38) homozygous for white flower colour were selected for high oil content (47%) and Line 15; a selection from these plants produced fertile hybrids with rape, broccoli and kale without embryo culture. In reciprocal crosses between oilseed rape cv. Tower and an aphid resistant diploid kale, 28 and 56 chromosome F₁ hybrid plants were regenerated from somatic embryos. The 56 chromosome plants were self-fertile and it was concluded from F₂ segregation ratios that a single dominant gene controls resistance to cabbage aphid in kale. The 28 chromosome F₁'s were self-sterile, but these and the 56 chromosome F₁'s could be backcrossed to rape and kale. A cross between the F₁ (2n56) and a forage rape resulted in the selection of a cabbage aphid (Brevicoryne brassicae L.) resistant line (Line 3). Both Line 15 and Line 3 can serve as bridges for gene interchange between B. campestris, B. napus and B. oleracea, which has not been possible hitherto. Hybridisations between rape and tetraploid kale produced F₁ plants with 37 chromosomes. One F₂ plant possessed coronal scales and the inheritance was shown to be controlled by a single recessive gene unlinked to petal colour.This paper is dedicated to Mr. T. P. Palmer, a colleague and close friend who retired from the DSIR as Assistant Director of the Crop Research Division in September 1984     

SEMISTERILITY IN THE INTERSPECIFIC HYBRID MELILOTUS POLONICA × M. ALBA

Jaranowski , J. K. (Coll. of Agriculture, ul. Wojska Polskiego 71c, Poznan, Poland.) Semisterility in the interspecific hybrid Melilotus polonica × M. alba. Amer. Jour. Bot. 48(1): 28–35. Illus. 1961.—Interspecific hybrids between Melilotus polonica (n = 8) and M. alba (n = 8) are readily secured. The F₁ hybrids are intermediate between the parents and partially sterile with a mean percentage of 58.8 (ranging from 46.8 to 72.6) defective pollen grains. Six bivalents and a chain or ring of 4 chromosomes occur at diakinesis and metaphase-I of microsporogenesis. A crossshaped configuration characteristic of a reciprocal translocation is present at pachytene, indicating that one of the parents is homozygous for an interchange of relatively large section between two of the members of the chromosome complex. Chromosome bridges, lagging chromosomes, movement of the univalents to the same pole and precocious division of the univalents lead to aberrant chromosome distribution during the course of meiosis. Reduction in self-fertility indicates a corresponding aberrant distribution of chromosomes during megasporogenesis. Pollen sterility in the F₂ generation ranged from 24.8% to 72.5% with a mean value of 54.6%. Two plants in the F₂ generation which had relatively low pollen sterility proved to be aneuploids (2n + 1). Meiotic irregularities in the F₂ plants were comparable to those exhibited by the F₁ plants.     

Intergeneric hybridization between Brassica napus and Sinapis pubescens,and the cytology and crossability of their progenies

The cytological possibility of gene transfer from Sinapis pubescens to Brassica napus was investigated. Intergeneric hybrids between Brassica napus (2n = 38) and Sinapis pubescens (2n = 18) were produced through ovary culture. The F₁ hybrids were dihaploid and the chromosome configurations were (0–1) _III + (2–11) _II + (5–24) _I . One F₂ plant with 38 chromosomes was obtained from open pollination of the F₁ hybrid. Thirty-one seeds were obtained from the backcross of the F₂ plant with B. napus. Five out of seven plants had 38 chromosomes, and the pollen stainability ranged from 0% to 81.4%. In the B₂ plants obtained from the backcross of B₁ plants with B. napus, 66.7% of the plants examined had 38 chromosomes. S. pubescens may become a gene source for the improvement of B. napus.     

Wide hybridization in Brassica

Summary Crosses were made to obtain interspecific hybrids between B. fruticulosa (wild species , 2n = 16) × B. campestris (cultivar , 2n = 20). Although many pollen grains germinated and their tubes entered the style, only about 30% of the ovules received pollen tubes. Fertilized ovules aborted at various stages of development. A few hybrid seeds resulted from hand pollinations in the field, and they showed poor germination and seedling establishment. The in vitro culture of ovaries, ovules, and seeds increased the frequency of obtaining hybrid seeds and plants: the most effective method was ovary culture followed by ovule culture. The hybrid nature of the plants was confirmed through morphological, cytological, and electrophoretic studies. A meiotic analysis of F₁ hybrids (2n = 18) showed that they had 0–5 bivalents and were completely pollen sterile. Electrophoretic analysis of leaf esterases and acid phosphatases of F₁ hybrids revealed bands derived from each parent. Induced amphidiploids of F₁ hybrids contained mostly bivalents, and had about 50% fertile pollen.     

Expression of Fertility during Morphogenesis in Self-Pollinated Backcrossed Progenies of Barley—Wheat Amphiploids [Hordeum geniculatum All. (2n = 28) × Triticum aestivum L. (2n = 42)] (2n = 70)

The fertility characteristics expressed during morphogenesis in first-generation self-pollinated backcrossed progenies (BC₁) obtained from amphiploid barley–wheat hybrids [Hordeum geniculatum All. (2n= 28) ×Triticum aestivum L. (2n= 42)] (2n = 70) backcrossed with common wheat were studied. It was found that, in the case of self-pollination of BC₁ plants, karyotype stabilization leads to the formation of alloplasmic euploid (2n = 42), telocentric substitution (2n = 40 + 2t), and telocentric addition (2n = 42 + t), (2n = 42 + 2t) plant forms, which may serve as the sources of the respective alloplasmic lines of common wheat. That the expression of fertility characters in BC₁F₈plants was shown to depend on growth conditions. The main mechanism of hybrid incompatibility of BC₁F₁–BC₁F₈plants was expressed as grass-clump dwarfism.     

Cytogenetic analysis of hybrids derived from wheat and <Emphasis Type="Italic">Tritipyrum</Emphasis> using conventional staining and genomic <Emphasis Type="Italic">in situ</Emphasis> hybridization

The new salt tolerant cereal, Tritipyrum (2n=6x=42, AABBE^bE^b) offers potential to introduce desirable characters for wheat improvements. This study was aimed to generate a segregating population from Iranian local wheat cultivars (2n=6x=42, AABBDD) and Tritipyrum crosses, study of the meiotic behaviour in F₂ hybrids and identification of E^b chromosomes in F₃ individuals. Results showed meiotic abnormalities in F₂ plants and different pairing frequency in the meiosis among F₂ plants. Genomic in situ hybridization revealed that total and E^b chromosome number of F₃ seeds ranged from 39 to 45 and 0 to 10, respectively. A significant prevalence of hyper-aneuploidy was observed among F₃ genotypes. C-banding patterns identified E^b chromosomes in Tritipyrum, indicating that it also can be useful to study wheat-Tritipyrum derivatives.     

Crossability of Brassica tournefortii and B. rapa, and morphology and cytology of their F1 hybrids

The crossability between Brassica tournefortii (TT, 2n = 20) and Brassica rapa (AA, 2n = 20) and the cytomorphology of their F₁ hybrids were studied. Hybrids between these two species were obtained only when B. tournefortii was involved as a female parent. The hybrid plants were intermediate for most of the morphological attributes and were found to be free from white rust under field conditions. The F₁ plants showed poor pollen fertility, although occasional seed set was achieved from open pollination. Self-pollination or backcrosses did not yield any seeds in these plants. The occurrence of chromosome association ranging from bivalents (0–7), trivalents (0–2) to a rare quadrivalent (0–1) in the dihaploid hybrids indicates pairing between the T and A genomes. The homoeologous pairing coupled with seed set in the F₁ plants offer an opportunity for interspecific gene transfers from B. tournefortii to B. rapa and vice-versa through interspecific hybridization. Received: 3 July 2000 / Accepted: 22 September 2000     

Barley Chromosome Identification Using Genomic in Situ Hybridization in the Genome of Backcrossed Progeny of Barley–Wheat Amphiploids [Hordeum geniculatum all. (2n = 28) × Triticum aestivum L. (2n = 42)] (2n = 70)

Genomic in situ hybridization (GISH) has been used to study characteristics of the formation of alloplasmic lines detected among self-pollinated backcrossed progeny (BC₁F₅–BC₁F₈) of barley–wheat amphiploids [Hordeum geniculatum All. (2n = 28) × Triticum aestivum L. (2n = 42)] (2n = 70). The chromosome material of the wild barley H. geniculatum has been shown to contribute to these lines. For example, fifth-generation plants (BC₁F₅) had genotypes (2n= 42w + 2g), (2n = 42w + 1g + 1tg), and (2n = 41w + 1g), where w is common wheat chromosomes, g is barley (H. geniculatum) chromosomes, and tg is the telocentric chromosome of wild barley. Beginning from theBC₁F₆ generation, alloplasmic telocentric addition lines (2n= 42 + 2tg) and (2n = 42 + 1tg) appear. This lines has been found cytogenetically unstable. The progeny of each of these cytological types include not only the (2n= 42 + 2tg) and (2n = 42 + 1tg) addition plants, but also plants with the monosomic (2n = 41 + 1tg) and the disomic (2n = 40 + 2tg) substitutions, as well as the (2n = 41 + 2tg) plants, which lack one wheat chromosome and have two telocentric barley chromosomes. It has been demonstrated that the selection for well-filled grains favors the segregation of telocentric addition lines (2n = 42 + 2tg) and (2n = 42 + 1tg).     

Specific Features of the Nuclear Genome Recombination in Backcross Progenies of Barley–Wheat Hybrids Hordeum vulgare L. (2n = 14) × Triticum aestivum L. (2n = 42)

The backcross progenies of the barley–wheat hybrids Hordeum vulgare L. (2n = 14) × Triticum aestivum L. (2n= 42) and two alloplasmic lines derived from them were studied using microsatellite markers of barley and wheat. The F₁ hybrids and first backcross plants BC₁ contained the genetic material of both cultivated barley and the cultivars of common wheat involved in developing of these hybrid genotypes. The genomes of BC₃, BC₄, and alloplasmic lines contained no microsatellite markers of the cultivated barley, whereas chromosomes of each homeologous group of common wheat were identified. In chromosomes of backcross progenies BC₃, BC₄, and alloplasmic lines yielded by backcrosses of hybrids and various common wheat cultivars, microsatellite markers of the parental wheat cultivars were shown to undergo recombination.     

CYTOTAXONOMY OF TWELVE SPECIES OF HIBISCUS SECTION FURCARIA

Menzel, Margaret Y. (Florida State U., Tallahassee), and F. D. Wilson. Cytotaxonomy of twelve species of Hibiscus section Furcaria. Amer. Jour. Bot. 50(3): 262–271. Illus. 1963.—Metaphase-I chromosome numbers and pairing in 88 accessions showed that H. cannabinus, H. costatus, and H. surattensis are diploid (n = 18); and H. acetosella, H. aculeatus, H. bifurcatus, H.furcellatus, H. meeusei, H. radiatus, H. rostellatus and H. sabdariffa are tetraploid (n = 36), with similar low multivalent frequencies, hence probably allotetraploids each combining 2 well-differentiated genomes. No intraspecific variation in ploidy was found. Fertile, vigorous F₁ hybrids between H.furcellatus and H. bifurcatus showed complete chromosome pairing (n = 36), confirming a close relationship between the parents. Two African strains of H. diversifolius were octoploid (n = 72) with low multivalent frequency and hence probably contain 4 differentiated genomes. At least 4, perhaps 5 or 6, differentiated genome groups are represented in tropical Africa, and at least 2 in the American tropics.     

Cytogenetic study of an interspecific cross of Nicotiana debneyi X N. umbratica

Summary Interspecific F₁ hybrids of Nicotiana debneyi Domin (2n=48) and N. umbratica Burbidge (2n=46), both belonging to the section Suaveolentes, showed a high degree of meiotic chromosome pairing. Two of the five F₂ plants obtained exhibited chromosome mosaicism. The first colchiploid generation (C₁) had the expected chromosome number of 2n=94 while C₂ showed 2n=88, a loss of three pairs of chromosomes. This same chromosome number continued in further colchiploid generations, followed up to C₅, except for a few plants in C₃ which showed chromosome mosaicism. The F₁ phenotype was stable through C₁ to C₅ and fertility was normal in colchiploids through all generations in spite of the loss of three pairs of chromosomes and chromosome mosaicism. This stability and fertility apparently reflect the tolerance of the genomes to the genetic adjustment of chromosome complements which is believed to be associated with the originally polyploid nature of the parental species and the chromosome doubling brought about in the amphidiploids.     

The effect of Secale cereale L. heterochromatin on wheat chromosome pairing

Metaphase-I chromosome association in PMCs of five F₁ hybrids 6x-triticale x T. turgidum (2n=5x=35 and genomes AABBR), and 13 plants from their backross or self offspring is reported. In wheat 18 chromosome arms and in rye 14 arms were recognized after C-banding and individually studied. Plants of backcross and F₂ showed variability for number and type of rye chromosomes, having in common the 28 durum wheat chromosomes (AABB). By testing meiotic association in plants with different rye chromosome constitutions, significant negative correlations were found. A clear negative effect of rye heterochromatin on pairing in wheat chromosomes is observed, the influence being more pronounced for large arms than for the short ones.     

INHERITANCE OF NUCLEAR 2C DNA CONTENT VARIATION IN INTRASPECIFIC AND INTERSPECIFIC HYBRIDS OF MICROSERIS (ASTERACEAE)

Nuclear DNA content varies over 20% within the diploid (2n = 18) species M. douglasii and M. bigelovii. Two different intraspecific crosses were made between M. douglasii biotypes which differed by about 10% in 2C nuclear DNA content. The F₂ progeny of one intraspecific cross showed no striking evidence of segregation for DNA content. The mean DNA contents of F₂ progeny from two sister hybrids from the second intraspecific cross were significantly different at the 1% level. An interspecific cross was made between biotypes of M. douglasii and M. bigelovii that differed by approximately 10% in DNA amount. The 12 F₁ progeny did not cluster around the parental midpoint, but instead encompassed nearly the entire range between the parental means. The five families of F₂ progeny studied each had a mean DNA content corresponding to that of the particular F₁ from which they were derived, indicating that the F₁ plants were not of identical DNA content. The results of this study suggest that DNA sequences which account for the DNA content differences among the plants are unstable and can undergo deletion or amplification in a hybrid. The altered DNA content may be heritably stable and show little or no segregation in the F₂ progeny.     

Interspecific hybridization of perennial Medicago species using ovule-embryo culture

Summary New interspecific hybrids between alfalfa (Medicago sativa L.) and several perennial Medicago species were obtained by embryo rescue techniques. The methodology, designated ovule-embryo culture, involved preculturing the fertilized ovule (10 to 20 days post-pollination) for a period of six to 12 days followed by excision and direct culture of the embryo. Placement of the hybrid embryo directly onto culture medium without the interim ovule culture was unsuccessful. Ovule culture to germination without removing the embryo also was unsuccessful. Ovule-embryo culture was essential for recovering interspecific hybrids between diploid alfalfa (2n=2x=16) and the following diploid (2n=2x=16) species: M. hybrida Traut., M. marina L., M. papillosa Boiss., M. rhodopea Velen. and M. rupestris M.B. In addition, trispecies hybrids between M. sativa x M. dzhawakhetica Bordz. F₁ hybrids (2n=3x=24) and either M. cancellata M.B. (2n=6x=48) or M. saxatilis M.B. (2n=6x=48) were obtained from ovuleembryo culture. Media manipulations using M. sativa x M. rupestris F₁ and first backcross generation embryos demonstrated the optimum concentration of 12.5 mM NH₄ ⁺ for successful embryo rescue; ammonium salt formulation (whether chloride, nitrate or sulfate) was not critical. From a few thousand crosses, hybrids between M. sativa and either M. rhodopea or M. rupestris were recovered relatively efficiently with 157 and 66 hybrids, respectively. However, only 13 hybrids between M. sativa and M. papillosa were obtained from more than 2,000 crosses, and just two hybrids each have been recovered from the combinations M. sativa x M. hybrida and M. sativa x M. marina from 2,000 to 3,000 crosses. The predominant chromosome number between diploid alfalfa and the other diploid perennial species was 2n=2x=16. Morphology of the hybrids was generally intermediate. Electrophoretic analysis of the F₁ hybrids and parental clones on uniform or gradient polyacrylamide gels demonstrated that peroxidase phenotypes could be used to confirm hybridity. For all interspecific combinations there was at least one peroxidase isozyme unique to the wild species that was present in the F₁ interspecific hybrid.