Overcoming hybridization barriers in potato

The cultivated potato is a major crop worldwide. It is a high input crop with complex quality requirements at harvest and during storage. Potato breeders are fortunate to have access to a very diverse and accessible germplasm resource. Wild Solanum relatives provide genetic diversity as well as genes for valuable production and quality traits. In most cases, crossing success can be predicted based on endosperm balance number (EBN), or effective ploidy, of the parents. Crossing barriers between most wild species and the cultivated potato are the consequence of differences in EBN and can be easily overcome using ploidy manipulations and bridge crosses. The most common ploidy manipulations include haploid extraction to reduce EBN and 2n gamete production to increase EBN. Additional methods to produce fertile interspecific hybrids include mentor pollination, embryo rescue, hormone treatments, reciprocal crosses, selection of cross‐compatible genotypes and somatic fusion. Knowledge of crossing barriers and mechanisms to overcome them allows potato breeders access to the rich gene pool in the genus Solanum.     

Interspecific hybridization of a multiploid mutant of durum wheat with rye and Triticum monococcum L. results in pentaploid hybrids

The multiploid mutant of durum wheat is a genotype that produces unreduced gametes. Our objective was to test the recovery of pentaploid hybrids in crosses of the mutant with rye and Triticum monococcum L. Compared with check crosses, the mutant had a two‐third reduction in percent seed set for rye crosses, but had only a slight decrease in crossability with T. monococcum. Pentaploid hybrids were associated with plump seeds of the mutant/rye cross, and with shrivelled seeds of the mutant/T. monococcum cross. We suggest that the endosperm balance number hypothesis explains the association of pentaploid hybrids with endosperm type. This association made for easy recovery of pentaploid hybrids from crosses to both species. Mature, plump seeds from the mutant/rye cross were germinated and pentaploid hybrids were recovered. One pentaploid hybrid was recovered for every 50.5 and 15.1 florets pollinated with rye and T. monococcum, respectively. Unreduced gametes in the multiploid mutant will facilitate interspecific hybridization by reducing the time to produce pentaploid plants.     

The importance of Endosperm Balance Number in potato breeding and the evolution of tuber-bearing Solanum species

Summary Endosperm failure is considered the primary reason for the lack of success in intra-and interspecific crosses. The Endosperm Balance Number (EBN) hypothesis is a unifying concept for predicting endosperm function in intraspecific, interploidy, and interspecific crosses. In the EBN system, every species has an effective ploidy (EBN), which must be in a 2:1 maternal to paternal ratio in the endosperm for crosses to succeed. The knowledge of EBN is very useful in the transfer of genes from exotic germplasm, and in the development of new breeding schemes in potato. The paper describes the strategies for introducing 2x(1EBN), 2x(2EBN), 4x(2EBN) and 6x(4EBN) germplasm into the cultivated 4x(4EBN) potato gene pool. A new methodology for producing 4x(4EBN) and 2x(2EBN) chromosome addition lines is also discussed. EBN has evolutionary importance in the origin of tuber-bearing Solanums. The role of the EBN in the origin of diploid and polyploid potato species, and as a barrier for hybridization and speciation of sympatric species within the same ploidy level is demonstrated. The origin of 3x and 5x cultivated tuber-bearing Solanums may also be explained using the EBN concept. EBN has been reported to exist in other plant species: alfalfa, beans, blueberries, rice, soybeans, squashes, tomato, forage legumes, grasses, ornamentals and Datura stramonium. This indicates that EBN may have broad application and could be useful for germplasm transfer and breeding in other crop species.     

Effects of <Emphasis Type="Italic">Solanum demissum</Emphasis> chromosomes on crossability in the backcross progeny to <Emphasis Type="Italic">Solanum tuberosum</Emphasis>

A balance of maternal and paternal genetic factors, conceptually named the endosperm balance number (EBN), is required for normal endosperm development in interspecific crosses in potato. We previously found that Solanum demissum (D), a hexaploid wild species widely used in potato breeding, has a slightly lower EBN than S. tuberosum (T). To explore the genetic nature of the EBN, the berry-setting rate, seed number/berry, and seed weight were evaluated in BC₁ [(D?×?T)?×?T] plants, each possessing different portions of the S. demissum chromosomes, by reciprocal crosses with D and T, and a quantitative trait locus (QTL) analysis was performed. At least 99 S. demissum-derived QTLs were detected, of which 29 were associated with differential responses to D and T. Three QTLs were possibly co-localized on chromosomes 7A and 10D1, while the remaining 23 QTLs were independently located. The QTLs in the three S. demissum homoeologous chromosomes exhibited three types of interaction: (1) positive, (2) negative, and (3) one positive and one negative effect on the same trait. We found that several major genes, one of which was localized in the S. demissum chromosome 9A, and many minor genes controlled the crossability of BC₁ plants. The QTLs responsible for the differential responses to D and T were different between the BC₁ plants used as male and female parents, indicating that different genes control the male and female EBNs. Consequently, we conclude that the EBN is represented by the sum of various genetic effects controlled by a large number of genes.     

Interspecific hybridization in sexual diploid Allium senescens var. minor× apomictic tetraploid A. nutans and sexual diploid A. senescens var. minor× apomictic hexaploid A. senescens

Fourteen interspecific hybrids in sexual diploid Allium senescens var. minor× apomictic tetraploid Allium nutans L. crosses, and eight interspecific hybrids in sexual diploid A. senescens var. minor× apomictic hexaploid A. senescens L. crosses were produced. The number of chromosomes was 2n= 24 in interspecific hybrids of diploid × tetraploid, and 2n= 32 in diploid × hexaploid crosses. Triploid and tetraploid interspecific hybrids showed intermediate parental morphological characteristics. Tetraploid interspecific hybrids of A. senescens var. minor×A. senescens crosses formed two groups based on leaf colour and leaf width. Seeds were formed in 11 out of 14 triploid interspecific hybrids under natural conditions. In cytological observations of parthenogenesis, three out of 12 triploid interspecific hybrids and five out of eight interspecific tetraploid hybrids were observed. Parthenogenesis ranged from 26.0% to 86.0% in five tetraploid interspecific hybrids. Non-parthenogenesis to parthenogenesis segregated in a 3:5 ratio in A. senescens var. minor×A. senescens crosses.     

Breakdown of a hybridization barrier between Solanum pinnatisectum Dunal and potato using the S locus inhibitor gene (Sli)

Several attempts have been undertaken to exploit the resistance to late blight [Phytophthora infestans (Mont.) de Bary] of the Mexican diploid Solanum pinnatisectum Dunal (2n = 2x = 24). However, sexual hybridization between S. pinnatisectum and cultivated potatoes is almost impossible because of strict reproductive barriers. In this study, we first induced chromosome doubling of S. pinnatisectum for matching of the species-specific endosperm balance number (EBN), which is a genetic phenomenon responsible for normal endosperm development. We pollinated 4x (2EBN) S. pinnatisectum flowers with pollen from 2x (2EBN) clones carrying the S locus inhibitor gene (Sli), which originally was identified as an inhibitor of S-locus-controlled self-incompatibility in pollen of diploid potatoes. Nine putative hybrid plants were obtained from 613 pollinations, of which two plants were characterized. Both individuals were triploid hybrids with extreme resistance to P. infestans derived from S. pinnatisectum and showed an acceptable level of female fertility for backcrossing. Thus, utilization of the Sli gene, combined with chromosome-doubling for matching of the EBN, is proposed as a technique to overcome barriers to interspecific hybridization between Mexican 2x (1EBN) Solanum species and cultivated diploid potatoes.     

Tolerance to low temperatures and tuber soft rot in hybrids between Solanum commersonii and Solanum tuberosum obtained through manipulation of ploidy and endosperm balance number (EBN)

The objectives of this study were to evaluate the tolerance to low temperatures and tuber soft rot in hybrids between Solanum commersonii and Solanum tuberosum. The experimental materials consisted of F₁ triploid, BC₁ pentaploid‐near pentaploid and BC₂ tetraploid–near tetraploid hybrids. The F₁ triploids had a freezing tolerance and acclimatization capacity closest to S. commersonii. This indicated that the endosperm barriers which prevent the introgression of 1EBN S. commersonii into 4EBN S. tuberosum had been overcome. Indeed, the triploids produced 2n eggs, thus giving a compatible maternal to paternal EBN ratio in the hybrid endosperm generated by the 3x(2EBN) × 4x(3EBN) crosses. The tolerance to low temperatures of BC₁ and BC₂ hybrids was lower than that of the F₁. However, a number of genotypes were identified which were able to withstand temperatures down to ‐5°C. Some BC₂ hybrids were also tested for their tolerance to tuber soft rot, and some resistant hybrids were detected. A number of them combined the capacity for cold acclimatization with tolerance to tuber soft rot. These hybrids have an EBN of 4; they are fertile and have been used in backcrosses with 4EBN S. tuberosum.     

Relevance of unilateral and bilateral sexual polyploidization in relation to intergenomic recombination and introgression in <Emphasis Type="Italic">Lilium</Emphasis> species hybrids

Sexual polyploids were induced in diploid (2n = 2x = 24) interspecific F1 hybrids of Longiflorum × Asiatic (LA) and Oriental × Asiatic (OA) Lilium hybrids by backcrossing to Asiatic (AA) parents as well as by sib-mating of the F1 LA hybrids. A majority of the BC1 progenies were triploid and the progenies from sib-mating were tetraploid or near tetraploids. Genomic in situ hybridization (GISH) technique was applied to assess the intergenomic recombination in the BC1 populations of LA and OA hybrids obtained after unilateral sexual polyploidization. A total of 63 LA (LA × AA and AA × LA) and 53 OA hybrids were analysed. LA hybrids were originated through the functioning of 2n gametes either as 2n eggs or 2n pollen while those of OA hybrids originated through functional 2n pollen of diploid OA genotype. In both type of crosses, a majority of the progenies had originated through First Division Restitution (FDR) mechanism of functional 2n gamete either with or without a cross over. However, there were nine LA- and four OA-genotypes where Indeterminate Meiotic Restitution (IMR) was the mechanism of 2n gamete formation. Based on GISH, total amount of introgression of Longiflorum and Oriental genome into Asiatic genome was determined. Most of the BC progenies exhibited recombination and the amount of recombination was higher in LA hybrids as compared to OA hybrids. Intergenomic recombination was also determined cytologically in the 16 plants of sib-mated LA hybrids where both parents had contributed 2n gametes. Based on these results the nature of interspecific lily hybrids obtained from uni- and bilateral sexual polyploidization leading to allotriploid and allotetraploid formation is discussed in the context of introgression and intergenomic recombination.     

Trifolium ambiguum as a source of variation in the breeding of white clover

Summary Hybrids between T. ambiguum and T. repens were successfully established after ovule culture. The hybrids were male sterile but one of the hybrids produced a single seed when backcrossed to T. repens, while the other produced a significantly greater number of seeds. The majority of the backcross hybrids from the latter had 48 chromosomes, being produced from the fertilisation of unreduced gametes of the F1 hybrid by T. repens pollen. Regular meiosis in the BC1 hybrid resulted in the production of balanced gametes containing the full complement of T. repens chromosomes and the polyhaploid set of T. ambiguum chromosomes. There was no difficulty in establishing a substantial population of BC2 hybrids. The meiotic behaviour of the BC1 hybrid indicated that it should be possible to transfer genes from T. ambiguum into white clover T. repens. The significance of the results in terms of the use of alien variation in the improvement of white clover is discussed.     

The transfer of the synaptic gene (sy-2) from 1EBN Solanum commersonii Dun. to 2EBN germplasm

Summary Crosses of a synaptic mutant (sy-2) of 2x(1EBN) S. commersonii Dun. with 2x(2EBN) S. chacoense Bitt. resulted in the production of 3x(2EBN) hybrids. Low levels of 2n egg production in these hybrids allowed subsequent crosses to be made with 4x(4EBN) cultivars, resulting in 5x(4EBN) S. commersonii-S. chacoense-Group Tuberosum hybrids. Seed set in these crosses averaged 0.22 seeds per fruit. Fifty-four unspotted seeds were produced in 1225 crosses of the 5x(4EBN) hybrids with 2x(2EBN) Group Phureja haploid extraction clones carrying dominant seed spot markers. These seeds yielded a total of 15 confirmed haploids having a diploid (2n=2x=24) or near diploid chromosome number. One haploid clone recovered displayed a synaptically abnormal phenotype similar to the original S. commersonii synaptic mutant. Preliminary test crosses of these haploids indicated that all haploids produced were 2EBN.This method for the transfer of alleles across EBN levels holds promise for the recovery of both qualitative and quantitative traits from 1EBN species. In these recovered, cross-compatible forms, clones with desired characteristics may be selected and easily incorporated into breeding programs.     

Morphological,cytological and reproductive characterization of tri-species hybrids (GOS) between <Emphasis Type="Italic">Pennisetum glaucum</Emphasis>, <Emphasis Type="Italic">P. orientale</Emphasis> and <Emphasis Type="Italic">P. squamulatum</Emphasis>

New tri-species hybrids (GOS) in the genus Pennisetum involving the cultivated species pearl millet (P. glaucum L.) and two wild species, viz. P. squamulatum Fresen and P. orientale L. C. Rich, are reported. Six hybrid plants were recovered after crossing a backcross hybrid (2n = 3x = 23, GGO) between P. glaucum (2n = 2x = 14, GG) and P. orientale (2n = 2x = 18, OO) with F₁s (2n = 6x = 42, GGSSSS) between P. glaucum (2n = 4x = 28, GGGG) and P. squamulatum (2n = 8x = 56, SSSSSSSS). The hybrids were perennial, morphologically intermediate to their parents, and represented characters from the three contributing species. The hybrids contained 2n = 44 chromosomes (GGGSSO) representing 21, 14 and nine chromosomes from P. glaucum, P. squamulatum and P. orientale, respectively. Meiotic and flow-cytometric analysis suggested origin of these hybrids from unreduced female and reduced male gametes. Average chromosome configuration (8.42_I + 14.32_II + 1.62_III + 0.52_IV) at Meiosis showed limited inter-genomic pairing indicating absence of significant homology between the three genomes. The hybrids were male sterile (except one) and highly aposporous. P. orientale was identified to induce apospory in hybrid background with P. glaucum at diploid and above levels, though it was quantitatively affected by genomic doses from sexual parent. A case of inducible and recurrent apospory is presented whereby a transition from Polygonum-type sexual embryo-sacs to Panicum-type aposporous embryo-sacs was observed in diploid interspecific hybrids. Results supported independent origin and partitioning of the three apomixis-components (apomeiosis, parthenogenesis, and functional endosperm development), reported for the first time in Pennisetum. Potential utilization of GOS hybrids in understanding genome interactions involved in complex traits, such as perenniality and apomixis, is discussed.     

Ploidy manipulation and introgression breeding in Darwin hybrid tulips

Meiotic polyploidisation via crossing with 2n gamete producing genotypes and interploidy crosses are two of the main methods currently used to obtain polyploid tulips. In our study diploid 2n gamete producing F₁ hybrids of Darwin hybrids (Tulipa gesneriana × Tulipa fosteriana) and triploid hybrid resulting from ‘Rhodos’ × ‘Princeps’ cross were used as pollen donor and crossed with cultivars of T. gesneriana in the following combination: 2x × 2x, 3x × 2x, 2x × 3x, and 3x × 3x. The progenies resulting from crosses at diploid level were mostly diploid, whereas a few seedlings were triploid. In 3x × 2x crosses aneuploids with chromosome constitution in between triploid and tetraploid (43–45 chromosomes) were predominant, but also one tetraploid (2n = 4x = 48) and four pentaploids (2n = 5x = 60) were obtained. In 2x × 3x crosses most progenies were triploid with the exception of a few aneuploids (3x + 1 and 3x ? 1), whereas in 3x × 3x cross diploid and aneuploid genotypes were recorded with chromosome number varied from 27 to 34. These results indicate that triploid parents produced aneuploid as well as euploid (x, 2x, 3x) gametes and that success in ploidy manipulation in tulip depends to a large degree on the ploidy level of the parental genotypes used for hybridization. Genome constitution of selected population of F1 and BC1 hybrids was analyzed through genomic in situ hybridization (GISH). GISH analysis of the BC1 showed a considerable amount of intergenomic recombination which is desirable for introgression breeding.     

The inheritance of a basal branching type in guar

Summary Eight different genotypes of the F₁ hybrids between Oriental × Asiatic lily (Lilium) hybrids (2n = 2x = 24) were treated with nitrous oxide (N₂O) gas under pressure for 24 and 48 hours. At the time of treatment, all plants possessed early meiotic stages in the anthers of the oldest flower buds. The mature flowers from treated plants were monitored for fertility through pollen germination in vitro as well as by using them in crosses with diploid Asiatic hybrids (2n = 2x = 24) both as male and female parents. In five out of the eight genotypes of OA hybrids there was evidence for the production of 2n pollen which germinated in vitro from either one or both treatments. The 2n pollen from three genotypes was successfully used in crosses. In two cases, the treated plants were successfully used as female parents which indicated the formation of 2n (or 2x) egg cells. From an analysis of 41 sexual polyploid progenies obtained from N₂O treated plants it was shown that they were all euploids consisting of 34 triploids (2n = 3x = 36) and seven tetraploids (2n = 4x = 48). A detailed cytological analysis of 12 progeny plants through genomic in situ hybridization (GISH) proved that N₂O had induced first division restitution gametes in most cases and in two cases they produced gametes through indeterminate meiotic restitution. There was evidence for intergenomic recombination in three cases.     

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.     

Interspecific hybridization in <Emphasis Type="Italic">Sarcococca</Emphasis> supported by analysis of ploidy level,genome size and genetic relationships

Knowledge of ploidy level differences, genome size and genetic relationships between species facilitates interspecific hybridization in ornamentals. For Sarcococca (Buxaceae) only limited (cyto)genetic information is available. The aim of this study was to determine the genome size and chromosome number and to unravel the genetic relationships of a breeder’s collection using AFLP marker analysis. Based on these results, interspecific crosses were made and the efficiency and hybrid status was verified. Two groups of diploid plants (2n = 2x = 24) were observed, with either a genome size of 4.11–4.20 or 7.25–9.63 pg/2C. All the tetraploid genotypes (2n = 4x = 48) had genome sizes ranging from 7.91 to 8.18 pg/2C. In crosses between parents with equal ploidy level and genome size a higher crossing efficiency (on average 58% of the hybridizations resulting in fruits) and more true hybrids (on average 96% of the offspring) were obtained compared to crosses between plants with different genome size and ploidy level (on average 23% fruits and 24% hybrids, respectively). In none of the cross combinations, the ploidy level or genome size was found to be a complete hybridization barrier, although unilateral incongruity was found in some cross combinations. Distant genetic relationships did not hamper the hybridization within Sarcococca genotypes. Our findings will contribute to a more efficient breeding program and a faster achievement of hybrids with an added value.     

Potential for analytic breeding in allopolyploids: an illustration from Longiflorum × Asiatic hybrid lilies (<Emphasis Type="Italic">Lilium</Emphasis>)

Ploidy level and intergenomic recombination was studied in interspecific hybrids between Longiflorum × Asiatic lilies (LA hybrid) backcross to Asiatic parents in order to assess the possibility for analytic breeding in lily. By backcrossing the diploid (2n = 2x = 24) F1 interspecific hybrid between Longiflorum × Asiatic lilies to Asiatic parents, 104 BC1 progeny plants were produced. Among these, there were 27 diploids, 73 triploids (2n = 2x = 36) and 4 aneuploids (2x − 1, 2x + 2 or 2x + 3). In addition, by backcrossing triploid BC1 (LAA) plants to diploid Asiatic parents in 2x − 3x and reciprocal combinations, 14 diploid BC2 progenies were produced. Genomic in situ hybridization (GISH) was performed to study the intergenomic recombination and karyotype composition. GISH indicated extensive intergenomic recombination among the chromosomes in LA hybrids. A large number of Longiflorum chromosomes were transmitted to the BC1 progenies from LA hybrids. However, very few Longiflorum chromosomes were transmitted from the BC1 triploid (LAA) plants to the BC2 progenies. The occurrence of diploid plants in the BC progenies of LA hybrids has opened the prospects of analytic breeding in lilies. In this approach, the selection of superior genotypes can be carried out at the diploid level and polyploid forms are synthesized from superior diploid parents. The advantages of analytic breeding are evident: (a) a maximum level of heterozygosity can be attained in the synthetic polyploids and (b) introgression can be achieved with a minimum of linkage drag. Based on GISH results the potential application of analytic breeding in lily allopolyploids has been discussed.     

Progenies of allotriploids of Oriental × Asiatic lilies (Lilium) examined by GISH analysis

With the aim of utilizing allotriploid (2n = 3x = 36) lily hybrids (Lilium) in introgression breeding, different types of crosses were made. First, using diploid Asiatic lilies (2n = 2x = 24), reciprocal crosses (3x − 2x and 2x − 3x) were made with allotriploid hybrids (AOA) obtained by backcrosses of F₁ Oriental × Asiatic hybrids (OA) to Asiatic cultivars (A). Secondly, the AOA allotriploids were crossed with allotetraploid (OAOA, 2n = 4x = 48), in 3x − 4x combination. Finally, the AOA allotriploids where crossed to 2n gamete producer F₁ OA hybrids (3x − 2x (2n)). Two types of triploids were used as parents in the different types of crosses, derived from: (a) mitotic polyploidization and (b) sexual polyploidization. Ploidy level of the progeny was determined by estimating the DNA values through flowcytometry as well as chromosome counting. The aneuploid progeny plants from 3x − 2x and reciprocal crosses had approximate diploid levels and in 3x − 4x crosses and 3x − 2x (2n) the progeny had approximate tetraploid levels. Balanced euploid gametes (x, 2x and 3x) were formed in the AOA genotypes. Recombinant chromosomes were found in the progenies of all crosses, except in the case of 2x − 3x crosses through genomic in situ hybridization (GISH) analyses. Recombinant chromosomes occurred in the F₁ OA hybrid when the triploid AOA hybrid was derived through sexual polyploidization, but not through mitotic polyploidization with two exceptions. Those recombinant chromosomes were transmitted to the progenies in variable frequencies.     

Interspecific compatibility barriers,development of interspecific hybrids through embryo rescue and lineage of Trifolium alexandrinum (Egyptian clover)—important tropical forage legume

Genetic improvement of Trifolium alexandrinum L. is hampered because of narrow genetic base and lack of interspecies compatibility information. The study was envisaged to understand the interspecies incompatibility and the affinity between T. alexandrinum and 22 species of the genus representing different sections and to develop interspecific crosses. Development of interspecific hybrids with T. resupinatum, T. lappaceum, T. subterraneum, T. vesiculosum and T. echinatum, through embryo rescue, revealed their affinity with T. alexandrinum. Failure of pollen germination or restricted pollen tube growth showed its distant relatedness with T. clypeatum, T. balansae, T. purpureum, T. leucanthum, T. hirtum, T. pilulare and T. hybridum. Pollen tubes reached up to ovule among the crosses with T. alpestre, T. repens and T. nigrescens, whereas it travelled up to ovary among the crosses with T. angustifolium, T. grandiflorum and T. dasyurum indicating need of manipulation to overcome barrier. Thus, incompatibility of T. alexandrinum with many Trifolium species could be overcome through embryo rescue with intensive crossing. Egyptian clover ecotype ‘Fahli’ was more compatible with other species than ‘Mescavi’.     

Pollination systems, hybridization barriers and meiotic chromosome behaviour in Nemesia hybrids

The ability of 13 Nemesia species (six annual and seven perennial) to sexually hybridize was investigated. Six of the perennial Nemesia species investigated were inter-fertile with one another. Two of the annual species, N. macroceras and N. strumosa, were inter-fertile. Thirty three crosses were successful and resulted in viable seeds. The analysis of meiotic chromosome behaviour in interspecific hybrids indicated that Nemesia chromosomes in different parental species were homeologous. No evidence of chromosome inversions or chromosome translocations was observed during meiosis in interspecific hybrids between the six perennial Nemesia species. In the hybrids produced between N. macroceras and N. strumosa, a quadrivalent was observed during meiotic metaphase I, indicating that these two species differ by a reciprocal translocation. A successful hybridization was made between N. anisocarpa (annual) and N. foetans (perennial), producing two triploid hybrids. In the unsuccessful crosses, pollen tubes were observed entering ovaries and ovules, suggesting that post-fertilization barriers were preventing sexual hybridization. Many of these crosses produced nonviable, shrunken, empty seeds, suggesting that endosperm breakdown and embryo abortion prevent interspecific hybridization in unsuccessful crosses. The manipulation of ploidy levels in N. fruticans and N. strumosa and tissue culture of N. strumosa × N. fruticans ovules failed to overcome post-fertilization barriers between these species.     

Rhizobium specificity of Trifolium ambiguum M. Bieb × T. repens L. hybrid clovers

Growth of full-sib families of an F₁ interspecific hybrid between Trifolium ambiguum M.Bieb and T. repens L, and two generations of backcross hybrids (BC₁F₂ and BC₂F₁) with T. repens as the recurrent parent, were compared to their parental species. Plants were grown in a N-free medium and inoculated with Rhizobium leguminosarum biovar trifolii rhizobia effective on T. ambiguum or T. repens. Hybridisation produced progeny that nodulated with rhizobia from either T. ambiguum or T. repens, but plant growth varied. Mean weights of T. repens and hybrids, particularly F₁ and BC₁F₂, were higher when inoculated with a mixture of strains isolated from field grown T. repens than with the New Zealand inoculant strain for T. repens. When inoculated with the mix of rhizobia from T. repens, mean weights were 242, 189, 132, 125, and 100 mg/plant for T. repens, BC₂F₁, BC₁F₂, F₁ and T. ambiguum, respectively. However, although the mean weight of BC₂F₁ lines was significantly less than T. repens, there was considerable variation in individual full-sib families indicating the potential to select within BC₂F₁ hybrids for high plant growth/symbiotic nitrogen fixation. The weight of T. ambiguumplants inoculated with the New Zealand inoculant strain for hexaploid T. ambiguum was similar to T. repens inoculated with the mix of rhizobia from T. repens (253 and 242 mg/plant, respectively). Mean fresh weights of F₁ hybrid plants were similar when inoculated with rhizobia for T. ambiguum or T. repens (125 and 130 mg/plant, respectively). However, weight of T. repens, BC₁F₂ and BC₂F₁ hybrids inoculated with rhizobia for T. ambiguum were all less than 90 mg/plant. There was a significant relationship between plant fresh weight and ethylene production. The results indicate that measuring weights of inoculated plants growing in N-free media is a rapid initial method of screening a range of plant germplasm for plant growth/symbiotic nitrogen fixation rates. This revised version was published online in August 2006 with corrections to the Cover Date.