Introgression of crown rust resistance from Festuca spp. into Lolium multiflorum

Crown rust, Puccinia coronata Corda, causes one of the most damaging foliar diseases in Italian ryegrass, Lolium multiflorum Lam. For introgression of crown rust resistance, highly resistant hybrids of the crosses Festuca arundinacea (2n = 6x = 42) ×L. multiflorum (2n = 4x = 28) and reciprocally, L. multiflorum (2n = 2x = 14) ×Festuca pratensis (2n = 4x = 28) and subsequently resistant recombinant individuals were used as female parents and susceptible cultivars of Italian ryegrass as male parents in three successive backcrosses. The BC₃ plants were selfed and crossed mutually. Uredospores of seven different crown rust isolates collected from plants of L. multiflorum, Lolium perenne, F. pratensis, F. arundinacea and L. multiflorum × F. pratensis hybrids were applied to identify the resistance or susceptibility of the parental species, backcrossed, selfed and intercrossed progenies. The various crown rust populations revealed a species-specific capability to infect plants of the Lolium-Festuca complex corresponding to the host species from which the spores originated. Selected BC⁹ plants, however, were found to be completely resistant to all crown rust populations tested. Successful introgression of the resistance was achieved from F. arundinacea as well as from F. pratensis. The resistance represents a dominant character, apparently based on a strong heterologous incompatibility between host and pathogen. In phenotype, bivalent formation during meiosis and in fertility, the novel germplasms are comparable with the L. multiflorum cultivars.     

The controlled introgression of Festuca arundinacea genes into Lolium multiflorum

Summary Using phosphoglucoisomerase (PGI/2) as a genetic marker, it has been shown to be possible to transfer genes from Festuca arundinacea into diploid Lolium multiflorum using the pentaploid hybrid L. multiflorum (4x) x F. arundinacea (6x). The pentaploid hybrid was sufficiently fertile to be used in reciprocal crosses with diploid. L. multiflorum. When used as the male parent, only two backcross generations were then required to reconstitute the diploid genotype. Intergeneric recombinants including a F. arundinacea PGI/2 allele were found among the diploid BC₂. Cytological data indicates that although the majority of chromosome associations involve only homologous Lolium chromosomes, associations involving Lolium and Festuca chromosomes also occur.Interpollinating the pentaploid hybrids prior to commencing a backcrossing programme increases the number of cycles of recombination and improves the chance of recovering intergeneric recombinants. The crossing programme described is proposed to be an effective method of introducing F. arundinacea genes into L. multiflorum.     

Unreduced gamete in interspecific hybrids in the Festuca/Lolium complex

Self-sterile interspecific hybrids between diploid Lolium perenne and either Festuca arundinacea or F. gigantea (both hexaploids) set some seeds when subjected to a heavy pollen cloud from the amphiploids derived from the hybrids. All the progeny were octoploid and were formed from the fertilization of unreduced gametes in the F₁ hybrid by amphiploid pollen. The significance of the results in relation to breeding efforts within the Lolium/Festuca complex is discussed.     

Androgenesis as a means of dissecting complex genetic and physiological controls: selecting useful gene combinations for breeding freezing tolerant grasses

The advantages of androgenesis from Lolium × Festuca hybrids as a means towards enhanced gene expression and capture of rare genetic variation are reviewed. New evidence is presented for the technique’s use in combination with introgression-mapping for targeting Festuca-derived genes for enhanced freezing-tolerance. As a starting point, a dihaploid genotype derived by androgenesis from a Lolium multiflorum × Festuca pratensis amphiploid (2n = 4x = 28) hybrid cultivar is used as female parent in a backcross breeding programme with L. multiflorum (2n = 2x = 14). A derivative of the backcross␣breeding programme was a genotype of L.␣multiflorum (2n = 2x = 14) incorporating a F.␣pratensis introgression on chromosome 4 that was more freezing-tolerant than Lolium. New evidence of the importance in Lolium and Festuca species of the adaptive capabilities of Photosystem II (PSII) in relation to subsequent freezing-tolerance, is presented. Non-photochemical quenching (NPQ) mechanisms for expulsion of excess light energy during cold acclimation are found in F. pratensis but not in L. multiflorum. Screens of a backcross population derived from an initial dihaploid genotype (n + n = 14) produced by androgenesis from a L. multiflorum × F.␣pratensis amphiploid, indicate a direct relationship between cold acclimation induced increases in NPQ and freezing-tolerance. Preliminary evidence of a role for genes found on chromosome 4 of F. pratensis for increased NPQ expression, is presented.     

Androgenesis from a Lolium multiflorum × Festuca arundinacea hybrid to generate extreme variation for freezing-tolerance

New opportunities for plant breeding using androgenesis in Lolium × Festuca hybrids have been identified. Plants derived by anther culture from a Lolium multiflorum × Festuca arundinacea (5x) hybrid were screened for freezing-tolerance, and their post-freezing recovery compared. The androgenic population showed extreme diversity in freezing-tolerance. While the majority of androgenic plants had inferior freezing-tolerance compared with the freezing-sensitive L. multiflorum parent, 6% of the population were more freezing resistant than the freezing-tolerant Festuca parent. Novel Lolium and Festuca gene combinations resulting from rare meiotic events were recovered within the androgenic population. The two most freezing-tolerant androgenic plants carried virtually the entire F. pratensis subgenome of F. arundinacea. F. pratensis is known to carry genes for freezing-tolerance and would be expected to be the primary source of genes governing this trait within the F. arundinacea genome. The most freezing-tolerant androgenic plants were more freezing-tolerant than the hybrid plant from which they were derived. Consequently, androgenesis was also effective in removing factor(s) reducing the expression of freezing-tolerance within the L. multiflorum × F. arundinacea (5x) hybrid.     

Cytogenetic Studies of Crosses between Lolium multiflorum Lam. and Festuca arundinacea Schreb. III. The Generations C1, C2 and C3

Meiotic observations of 56-chromosome plants of the C₁, C₂ and C₃ generations of the amphidiploid Lolium multiflorum × Festuca arundinacea showed that most of the chromosomes paired as bivalents. However, uni- and multivalents were frequently observed. No difference in meiotic regularity and stability could be detected between the different generations. The average chromosome number and; the percentage of 56-chromosome plants decreased with increasing generations. The direct use of the 56-chromosome amphidiploids of L. multiflorum and F. arundinacea in a breeding programme appears to be quite difficult, but these plants can be used in a backcross programme with Lolium and Festuca.     

Cytoplasmic male sterility in ryegrasses (Lolium SPP.) detected after intergeneric hybridization

Summary Among diploid derivatives of a tetraploid intergeneric hybrid originating from a cross between a diploid F₄ hybrid of Lolium perenne x L. multiflorum and an autotetraploid plant of Festuca pratensis as female and male parent, respectively, a great number of male-sterile Lolium plants have been found. The male sterility appears to be based on an interaction of one or two recessive nuclear genes and sterilizing cytoplasm. The data available indicate a high frequency of B types (O types) in perennial ryegrass.     

First in vivo assessment of feeding value of Festulolium hybrids derived from Festuca arundinacea var. glaucescens and selection for palatability

This paper reports on the first results of feeding value experiments, assessed with sheep, of hybrids between tetraploid Italian ryegrass (Lolium multiflorum, 2n = 4×= 28) and a progenitor of tall fescue, Festuca arundinacea var. glaucescens (2n = 4×= 28). Three hybrid populations resulting from either high, low or no selection for palatability were compared with a variety of Italian ryegrass as control and two varieties of the cultivated hexaploid (2n = 6×= 42) tall fescue, one of which had been selected for high palatability. On average, the hybrid populations were found to be as palatable as the improved tall fescue with voluntary intake (VI), in vivo digestibility of organic matter (DOM), and net energy expressed in fodder units for milk, similar to the best palatable tall fescue and to Italian ryegrass. However, selection for improved palatability among hybrids did not result in any further improvement of other traits, except VI in the hybrid selected for high palatability which significantly exceeded VI of Italian ryegrass. In conclusion, intergeneric hybridization between L. multiflorum and F. arundinacea var. glaucescens led to an immediate increase of feeding value compared with selection for palatability in tall fescue. However, to improve feeding value of hybrids further, similar selection for high palatability appeared questionable and possibly detrimental for other agronomic traits because of complicated inheritance in tetraploid L. multiflorum×F. glaucescens hybrids.     

Genomic constitution of Festulolium cultivars released in the Czech Republic

Genomic in situ hybridization (GISH) was used to characterize the chromosome constitutions of individual plants from a set of tetraploid and hexaploid cultivars of Festulolium developed and released in the Czech Republic from hybrids of Lolium multiflorum with Festuca pratensis and F. arundinacea. A simplified GISH protocol readily discriminated parental genomes in the hybrids and facilitated the screening of large numbers of plants per accession. The contribution of parental genomes in the cultivars tested ranged from predominance of chromatin from one of the parents to a more balanced contribution from both parents. However, in none of the cultivars were equal proportions of chromatin from both parents present. The parental contribution to the hybrids was both in the form of complete chromosomes or as chromosome translocations. In hexaploid cultivars from (L. multiflorum × F. arundinacea) × F. arundinacea hybrids the average numbers of complete L. multiflorum chromosomes ranged from 4.95 to 7.5 and the numbers of translocations from 6.33 to 10.21. Two tetraploid cultivars from (L. multiflorum × F. arundinacea) × L. multiflorum hybrids showed a strong prevalence of L. multiflorum chromatin and intergeneric translocations were rare. In the tetraploid cultivar ‘Perun’ of the L. multiflorum × F. pratensis hybrid there were 11.7 chromosomes of L. multiflorum and 14.7 recombined chromosomes on average. Reasons for the domination of one of the parental genomes in hybrid cultivars are not clear and are only partially explained by breeding history. Recombination rates of individual genomes in hybrids involving F. arundinacea were evaluated in double hybridization experiments. The results indicated a strong affinity of the L. multiflorum genome for the F. pratensis genome present in F. arundinacea and little affinity for the F. glaucescens genome. This suggests that introgressions from F. arundinacea into L. multiflorum are primarily limited to the F. pratensis genome which can be more readily accessed in L. multiflorum × F. pratensis hybrids.     

Introgression of Genetic Information from Triticum monococcum L, into Hexaploid Triticale by Hybridization with a T. monococcum×S. cereale Amphiploid

Four hexaploid triticale lines were crossed as females with a T. monococcum×S. cereale amphiploid (A^mA^mRR), with the aim of introducing the genetic material of diploid wheat. F₁-plants (A^mABRR)were back-crossed with a parental form of 6×-triticale as male and progenies were subjected to four different types of pollination with the aim of finding the optimal one in respect to gradual stabilization of introgressive hexaploid karyotypes. Beginning with BC₁-plants, a strong tendency to decrease the somatic chromosome number was observed. In subsequent generations this was accompanied by the decrease of seed germination and plant fertility. Both of these characters showed statistically significant dependence on somatic chromosome number variation which was analyzed in BC₁/F₂ and BC₂ populations. Based on spike fertility, an effective selection pressure was applied to restitute the hexaploid chromosome number. In the BC₁/F₄ generation, the first morphologically uniform secondary hexaploid lines were selected. 11.4% of the lines showed a non-waxy spike — a morphological marker transmitted from T. monococcum.     

Breeding for hard red winter wheat cultivars adapted to conventional-till and no-till systems in northern latitudes

Summary Monosomic alien addition lines combining individual F. drymeja chromosomes and the L. multiflorum complement were isolated from the cross between the triploid hybrid L. multiflorum (4x) × F. drymeja (2x) and diploid L. multiflorum (2x). Chromosome pairing in the addition lines was studied at metaphase 1 of meiosis and the relationship between single F. drymeja chromosomes and the corresponding homologous pair in L. multiflorum is discussed. Trivalent frequency in the addition lines was higher than expected from observations of chromosome pairing in the triploid hybrid and there were differences between lines in the number of trivalent associations formed. There is some evidence to suggest that trivalent frequency is not entirely dependent on chromosome length and that transmission of the alien chromosome in the female is dependent on the size of the added chromosome. Morphological studies were made to assess the phenotypic effects of the addition of single F. drymeja chromosomes to the L. multiflorum complement. Two plants (2n=14) with recombination between a L. multiflorum and a F. drymeja chromosome were identified.     

Inflorescence production in an amphiploid between Lolium multiflorum (2n=28) and Festuca arundinacea (2n=84)

Summary The flowering characteristics of an amphiploid Lolium multiflorum (2n=28)× Festuca arundinacea (2n=84) have been compared with those of the parental species under field and glasshouse conditions. In field drills the number of inflorescences produced by an unselected amphiploid population was markedly less than that produced by S.170 tall fescue and S.22 Italian ryegrass. Experiments to determine the requirements for floral induction in the amphiploid showed that they were intermediate between those of the two parents. There was no indication that the hybrid was not becoming induced under field conditions and it is concluded that the competitive condition in field drills was preventing floral initiation and development. A comparison of seed from third and fourth generation stock suggested that additional multiplication through a number of generations, and subsequently growing the derived basic seed at high nitrogen levels would improve inflorescence production in the amphiploid.     

The transfer of triazine resistance from Brassica napus L. to B. oleracea L. III. First backcross to parental species

Summary Atrazine resistant Brassica napus × B. oleracea F₁ hybrids were backcrossed to both parental species. The backcrosses to B. napus produced seeds in both directions but results were much better when the F₁ hybrid was the pollen parent. Backcrosses to B. oleracea failed completely but BC₁s were rescued by embryo culture both from a tetraploid hybrid (2n = 4x = 37; A₁C₁CC) and sesquidiploid hybrids (2n = 3x = 8; A₁C₁C). Progeny of crosses between the tetraploid hybrid and B. oleracea had between 25 and 28 chromosomes. That of crosses between the sesquidiploid hybrid and B. oleracea had between 21 and 27. A few plants that had chromosome counts outside the expected range may have originated from either diploid parthenogenesis, unreduced gametes or spontaneous chromosome doubling during in vitro culture. Pollen stainability of the BC₁s ranged from 0% to 91.5%. All the BC₁s to B. oleracea were resistant to atrazine.     

Infection of Lolium and Festuca spp. by Drechslera siccans and D. Catenaria

Summary Drechslera siccans was the commonest of the species of this leaf spot pathogen found on single spaced plants of Lolium perenne at Aberystwyth. D. catenaria and D. dictyoides were also frequently isolated. A wide range of genotypes within the host species L. perenne, L. multiflorum, Festuca arundinacea and F. pratensis, and structural hybrids between F. arundinacea and L. multiflorum, were all equally susceptible to conidial infection by D. siccans in the glasshouse and showed similar development of leaf spot symptoms, even though Festuca spp. are not normally attacked in the field. However, when exposed to infection out of doors there was significant variation in resistance, both between species and often also between genotypes within the same species. As in so many other instances of fungal infection, tetraploidy appeared to confer a higher level of resistance. The degree of resistance of a F. arundinacea × L. multiflorum amphiploid and two succeeding backcross generations to L. multiflorum approximated to that of the more susceptible L. multiflorum parent. By contrast to D. siccans, when plants were inoculated with D. catenaria in the glasshouse, both intervarietal and intravarietal variation in leaf spot development was evident. Resistance to D. siccans appears to depend partly on a limitation of conidial production, and to D. catenaria on restriction of hyphal growth following infection, which may explain the relative discrepancies between field and glasshouse results. The implications for resistance breeding are discussed.     

Increasing biodiversity of irrigated rice in Africa by interspecific crossing of Oryza glaberrima (Steud.) × O. sativa indica (L.)

The African rice Oryza glaberrima, traditionally cultivated since more than 3.500 years, is of poor agronomic performance but resistant/tolerant to various stresses and diseases. The introduction of these characters into O. sativa cultivars is difficult since crossing barriers cause spikelet sterility in F1. Backcrossing can restore fertility and recently facilitated the development of fertile O. glaberrima × O. sativa ssp. japonica hybrid progenies for rain fed systems. With the objective to gain access to African rice germplasm for improvement of irrigated rice, crosses were performed with eighteen O. glaberrima and twenty O. sativa ssp. indica accessions. In total about one hundred F1-hybrid grains were obtained. The F1 plants were all completely sterile and backcrossing (BC) to O. sativa was performed in order to restore spikelet fertility. Monitoring of Tog5681 × IR64 hybrid progenies under field conditions revealed a broad genetic diversity within the BC₁ and BC₂ populations. Some BC₁ and BC₂ progeny plants outperformed the O. sativa parent, indicating that the heterozygocity level and complementary gene action after two backcrosses are still sufficient to positively influence plant vigor. Spikelet fertility of progenies was highly variable, but almost complete fertility was already observed within the BC₁F₂ population. High spikelet fertility was preserved in one out of two analyzed BC₁F₃ families and inmost of the BC₂F₃ families. The ability to restore spikelet fertility within few generations and the potential of the genetic diversity present in interspecific progenies facilitates the development of plant types specifically designed for the African irrigated and lowland environment. This revised version was published online in August 2006 with corrections to the Cover Date.     

Cytological and Isozyme Evaluation of Tall Fescue × Italian Ryegrass Hybrids

Development of tall fescue (Festuca arundinacea Schreb., 2n = 6x - 42) × Italian rye-grass (Lolium multiflorum Lam., 2n = 2 ×= 14) hybrids would enhance efforts to improve the quality of tall fescue. Two ‘Kentucky 31’ tall fescue בLemtal Italian ryegrass hybrids were obtained via embryo rescue on MS media containing casein hydrolysate, ascorbic acid and sucrose. Chromosome pairing at metaphase I had an average of more than 12 bivalents per cell. Since Festuca-Lolium pairing can account only for seven of the paired chromosomes, intergenomic as well as interspecific chromosome pairing is indicated. There was no cytoplasmic effect on chromosome pairing. To determine if enzymes could be used as genetic markers for distinguishing hybrids from self-contaminants in crosses, zymograms of PGI, 6-PGD, MDH, GOT and ACPH were obtained from parents and hybrids using starch gel etectrophoresis. PGI, 6-PGD and MDH had fewer bands in the diploid ryegrass, as compared with the hexaploid tall fescue and the tetraploid hybrid.     

Assessing success in gene transfer betweenLolium multiflorum andFestuca arundinacea

Summary AFestuca arundinacea (2n=6x-42) plant with three PGI/2 homoeoalleles marking three homoeologous chromosomes was crossed with aLolium multiflorum plant (2n=4x=28) with a different PGI/2 phenotype to give a pentaploid hybrid (2n=5x=35) with five chromosomes each marked by a different PGI/2 allele. This hybrid plant was backcrossed twice with diploidL. multiflorum (2n=2x=14) with a different PGI/2 phenotype. Numbers of interspecific recombinants involving chromosomes marked by PGI/2 were then determined in both backcross generations. In the BC1, recombinants involving only one PGI/2 allele were found but in the BC2, all three homoeologousF. arundinacea chromosomes carrying the PGI/2 locus recombined withLolium with one in greater frequency and the others in equal but lower frequency. The evidence supports claims made forF pratensis (2n=2x=14) andF. arundinacea var.glaucescens (2n=4x=28) being progenitors forF. arundinacea.     

Chromosomal Stability in the Backcross Progenies of Pentaploid Hybrids between Avena sativa L. and A. maroccana Gdgr.

In a backcrossing programme to transfer desirable characters from wild Avena maroccana Gdgr. to cultivated oats, A. sativa L., meiotically stable plants in BC₁F₃ and BC₂F₂ progenies were isolated. The recovery of stable genotypes with 2n = 6×= 42 chromosomes indicated that two backcrosses are enough for such a programme. The cytological observations in various backcross generations are presented and discussed.     

Culture of embryos and shoot tips for chromosome doubling in Lolium perenne and sterile hybrids between Lolium and Festuca

An improved method is reported for polyploid induction in Lolium (ryegrass), and in sterile F₁ hybrids between Lolium and Festuca (fescue). Two factors greatly increased the survival rate of colchicine‐treated embryos of Lolium perenne (perennial ryegrass) germinated and cultured in vitro (1) a high concentration of sucrose (100 g/1) in the germination medium and (1), maintenance at a low temperature of 10°C for 2 weeks after treatment. The maximum success rate for chromosome doubling among survivors of perennial ryegrass was 79.1%, and for L. perenne×Festuca arundinacea F₁ hybrid embryos it exceeded 40%. The same doubling treatment also works with shoot tip culture in non‐ flowering genotypes obtained by anther culture of L. multiflorum×F. arundinacea hybrids.     

Production of an interspecific hybrid between Brassica fruticulosa and B. rapa

An interspecific hybrid between a wild species, Brassica fruticulosa (2n = 16, FF) and a crop Brassica species, B. rapa (2n = 20, AA) was synthesized using sequential ovary‐ovule culture on MS medium supplemented with casein hydrolysate. Morphological, molecular and cytogenetic analysis confirmed the true hybrid nature of the offspring. The F1 plants (2n = 18) were intermediate in morphology, highly pollen‐sterile as well as self‐sterile. A maximum of three bivalents per PMC was recorded, but 14 I + 2 II was the most common meiotic configuration. Normal pollen fertility and regular bivalent (18 II) formation was observed in the amphiploid sectors of the hybrid plants. The F₁ hybrid harboured significantly lower aphid populations than the crop Brassica parent.