The production and behaviour of hexaploid hybrids between Hordeum vulgare and H. Bulbosum

Summary To produce hexaploid (or other polyploid) hybrids, diploid or tetraploid Hordeum vulgare was crossed with hexaploid or octoploid H. bulbosum, and perennial triploid hybrids between the two species were treated with colchicine. The crosses did not yield viable plants: seedset was low, the seed aborted and embryo culture was unsuccessful. The colchicine treatments geve rise to plants in which hexaploid chromosome numbers were observed. At the hexaploid level chromosomal instability occurred, resulting in chromosome elimination.The colchicine-treated triploid hybrids showed in the first years after the treatment better fertility after open flowering than untreated plants, but the level of fertility remained very low. The offspring consisted of haploid, diploid and approximately triploid plants like H. vulgare, tetraploid and approximately tetraploid plants like H. bulbosum, and plants with hybrid morphology and unstable chromosome number, which were highly sterile. Thus the crossing barrier between H. vulgare and H. bulbosum could not be broken down at higher ploidy level.     

Characterization for agronomic use of cytoplasmic male-sterility in sunflower (Helianthus annuus L.) introduced from H. resinosus Small

A backcrossing programme was carried out both to assess the stability of a cytoplasmic male‐sterility (CMS) source from Helianthus resinosus, designated RES1, and to incorporate it into inbred sunflower lines (HA89, RHA271, RHA801). All the progenies, grown in different environments, were completely male‐sterile. This suggests that the expression of this cytoplasm is stable. Female‐fertility of lines HA89, RHA271 and RHA801 carrying CMS RES1 were compared with those of the corresponding fertile inbred lines. There were no differences in the number of seeds per head. This indicates that female‐fertility is not affected by RES1 cytoplasm. Cytological studies showed that meiosis proceeds normally until the tetrad stage; consequently, the absence of pollen is caused by alterations that take place during postmeiotic stages. With the aim of identifying male‐fertility restorer genotypes, crosses were made between HA89 (CMS RES1) plants and different annual diploid and perennial hexaploid Helianthus species. All the diploid germplasm evaluated behaved as a CMS RES1 maintainer. However, the hexaploid species, H. resinosus, H. x laetiflorus, H. pauciflorus and H. tuberosus, restored pollen fertility in CMS RES1 plants.     

Interspecific hybrid of Helianthus annuus × H. simulans: Characterization and utilization in improvement of cultivated sunflower (H. annuus L.)

The first success at interspecific hybridization between cultivated sunflower(H. annuus) and a diploid perennial species, H. simulans is reported. The F₁s from both direct and reciprocal directions exhibited dominance of the wild species phenotype and were pollen sterile. Meiosis was irregular in the F₁ hybrids and both univalents and multivalents were observed. Multiplication of the interspecific hybrids was achieved through in vitro culture of nodal sections on Murashige & Skoog medium supplemented with 0.5 mg l^-1 benzyladenine. Fertility of the interspecific hybrids was improved by subjecting the in vitro proliferating shoots to 0.001% colchicine incorporated in the shoot multiplication medium. The amphiploids serve as fertile bridges and facilitate interspecific gene transfer through conventional breeding. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

The resistance of Hordeum bulbosum and its hybrids with H. vulgare to common fungal pathogens

Summary Two tetraploid and two diploid clones of Hordeum bulbosum were screened for resistance to five isolates of powdery mildew which are virulent on cultivated barley. All were resistant and this resistance was also expressed in hybrids with H. vulgare. The tetraploid genotypes were also resistant to isolates of yellow rust and brown rust. These results show that H. bulbosum contains useful genes for resistance to these diseases and that there is a potential to transfer these into cultivated barley.     

Anther Culture-Derived Regenerants from Hordeum vulgare × H. bulbosum Crosses

Hordeum bulbosum has several desirable attributes, including disease resistance, which would be worthwhile transferring to H. vulgare. Despite homoeologous chromosome pairing in the interspecific hybrids, there have been few reports of successful gene introgression between the two species. A possible explanation for this is that recombinant male gametes are at a competitive disadvantage with normal balanced gametes during post-pollination events. To circumvent this problem, the possibility of obtaining plants directly from immature pollen grains was investigated. Anthers from diploid, triploid and tetraploid H. vulgare × H. bulbosum hybrids were cultured on defined media. Only hybrids with dehiscent anthers in vivo responded in culture, and after transfer of calli and embryoids to regeneration medium, 36 albino and 12 green plants were obtained. Seven of the green regenerants survived, one of which contained 15 H. vulgare chromosomes (including one acrocentric chromosome) and one H. bulbosum chromosome. Another regenerant (Ac166) resembled a diploid H. vulgare × H. bulbosum hybrid but had partial anther dehiscence and a slightly modified chromosome constitution. Mostly normal H. vulgare progeny were obtained from crosses between H. vulgare cv.‘Emir’ and Ac166, but three plants involved chromosome additions and substitutions.     

Discovery of 2n gametes in tetraploid oat Avena vaviloviana

Summary Sexual polyploidization via the action of 2n gametes (gametes with the sporophytic chromosome number) has been identified as the most important evolutionary mode of polyploidization among plant genera. This study was conducted to determine whether 2n gametes are present in the tetraploid level of the genus Avena (2n=4×=28) Twenty tetraploid Avena lines, representing four species and one interspecific hybrid, were screened for pollen grain size in order to differentiate between n and 2n pollen. Avena vaviloviana (Malz.) Mordv. line PI 412767 was observed to contain large pollen grains at a 1.0% frequency. Cytogenetic analyses of pollen mother cells of PI 412767 revealed cells with double the normal chromosome number (i.e., 56 chromosomes at metaphase I and anaphase I). The mode of chromosome doubling was found to be failure of cell wall formation during the last mitotic division that preceded meiosis. The resulting binucleate cells underwent normal meiotic divisions and formed pollen grains with 28 chromosomes. Based on the formation and function of 2n gametes, three models involving diploid and tetraploid oat lines are proposed to describe possible evolutionary pathways for hexaploid oats. If stable synthetic hexaploid oat lines could be developed by utilizing 2n gametes from diploid and tetraploid oat species through bilateral sexual polyploidization, the resulting hexaploids could be used in breeding programs for transferring genes from diploids and tetraploids to cultivated hexaploids.     

Hybridization between diploid (Gossypium arboreum) and tetraploid (Gossypium hirsutum) cotton through ovule culture

Summary With in vitro culture of ovules, interspecific hybrids have been obtained in an otherwise incompatible cross between a diploid (Gossypium arboreum) and a tetraploid (G. hirsutum) cultivated cotton. The early abortion of the embryo was prevented by repeated treatment of the flowers, immediately after pollination with a solution of gibberellic acid and naphthalene acetic acid. The ovules excised three days after pollination and cultured in a liquid medium underwent profuse proliferation, whereas on an agar-solidified medium supplemented with casein hydrolysate, indoleacetic acid and kinetin they germinated to form hybrid plants.     

Polyploidy and sexual polyploidization in the genus Vaccinium

Genus Vaccinium,consisting of blueberries, cranberries, lingonberries and many related wild species, includes diploid, tetraploid, and hexaploid species. Most evidence indicates that the tetraploid species are autotetraploids, with non-preferential bivalent chromosome pairing. Although homoploid interspecific crosses usually produce numerous fertile hybrids if the parents are from the same section of the genus, inter sectional crosses at the diploid level normally produce no seedlings, weak seedlings, or seedlings that are very low in fertility. There is a strong but not complete triploid block within Vaccinium. Even with insections, tetraploid × diploid (and the reciprocal) crosses normally give only a few tetraploid and a few triploid hybrids. Hexaploid × diploid crosses within sections are very hard to make, and the few hybrids that have been obtained are pentaploid. The frequency of 2n gametes varies,both among genotypes within species and among species. Vaccinium pollen is normally shed in tetrads, and the frequency of large pollen grains shed in dyads can be used to estimate 2n gamete frequency. Cultivated blueberries occur at both the tetraploid and the hexaploid levels, and there are important genetic resources in the diploids. Unreduced gamete production has permitted transfer of genetic material from the diploid to the tetraploid level and from the diploids and tetraploids to the hexaploid level via triploid hybrids. Intersectional crosses can occasionally produce tetraploid Vaccinium hybrids that appear to behave as amphidiploids and have medium to high fertility. CommercialVaccinium cultivars are normally propagated by cuttings. Intersectional hybridization, chromosome doubling, and asexual propagation could permit the production of novel hybrid combinations with value as ornamentals or in fruit production. This revised version was published online in July 2006 with corrections to the Cover Date.     

Isolation and characterisation of somatic hybrids of diploid Solanum tuberosum and Solanum brevidens and the use of amylose-free starch mutation for detection of introgression

Summary Somatic hybrids of diploid amylose-free (amf) Solanum tuberosum and diploid S. brevidens were made by Poly-Ethylene-Glycol (PEG) or electrofusion methods. For the isolation of interspecific hybrids the use of selection markers (kanamycin and hygromycin resistance) was useful but not essential. In this 2x+2x interspecific combination 4x and 6x somatic hybrids were obtained. Seed set was the best in 4x×4x (S. tuberosum) backcrosses, but seed germination was the best in 6x×4x combinations, using in vitro germination of unripe seeds harvested 25 days after pollination. A high degree of pollen stainability (30–40%) was observed in 7 tetraploid hybrids and very low in all hexaploids. After iodine staining, the recessive amf marker was expressed by a red colour instead of blue, visible in starch-containing cells like columella cells of root tips, (micro)tubers or microspores. As expected, complementation was observed in starch-containing cells of the fusion hybrids. Segregation of the amf marker was clearly observed in microspores of 4x and 6x hybrids. Segregation ratios in the 4x hybrids showed variable recombination frequencies. In the backcross progeny of hexaploid F12-5 with a tetraploid amf mutant one amylose-free recombinant among 67 plants was found, indicating the occurrence of meiotic recombination in the megaspore mother cells.     

Identification of somatic hybrids of dihaploid Solanum tuberosum lines and S. brevidens by species specific RAPD patterns and assessment of disease resistance of the hybrids

Summary Symmetric somatic hybrids were produced by electrofusion of protoplasts of two dihaploid tuber-bearing potato (Solanum tuberosum L.) lines and Solanum brevidens Phil., a diploid non-tuber-bearing wild potato species. A total of 985 plants was obtained. Verification of nuclear hybridity of putative hybrids was based on additive RAPD patterns, general morphological characteristics and chromosome counts. 53 (90%) calli regenerated into plants which were identified as somatic hybrids. Most of the hybrids were aneuploids at the tetraploid (4×) or hexaploid (6×) level. The 20 hybrids tested expressed a high level of resistance to potato virus Y (PVY ^N ) characteristic of the S. brevidens parent. Resistance to late blight (Phytophthora infestans (Mont.) de Bary) varied between hybrids, but was on average better than that of the fusion parents. Resistance of hybrids to bacterial stem rot (Erwinia carotovora subsp. atroseptica (van Hall) Dye) was not superior to that of commercial potato cultivars.     

Variation in progenies of an Arachis hypogaea x diploid wild species hybrid

Summary Derivatives of a cross between cultivated peanuts, Arachis hypogaea L. (2n=40), and the wild species collection GKP 10017 (2n=20) were compared morphologically, for leafspot resistance and for yield. The objective of the study was to determine the effects of wild species germplasm on the A. hypogaea genome. The sterile F₁ hybrid which resulted from crossing the two species was treated with colchicine to restore fertility at the 6x ploidy level. The resulting hexaploid was cytologically unstable and progeny lost chromosomes until stability was regained at the 2n=40 chromosome level. Forty-seven characters were used to analyze the variation among plants in the tetraploid interspecific hybrid population. The plants were compared to four cultivated lines plus GKP 10017. Many hybrids were intermediate to the two parents in morphology. Individual traits such as growth habit, pod and seed size, elongation of the constricted area between pods, nodulation and leaflet size were altered by the presence of GKP 10017 germplasm in many of the hybrid plants. Cercospora arachidicola Hori and Cercosporidium personatum (Berk. & Curt.) Deighton resistances were evaluated for all plants. Several hybrids had few lesions due to either leafspot pathogen. In addition, 24 largeseeded interspecific hybrid selections were compared to the cultivated variety NC 5 for yield. Five selections were superior to both parents at p=0.01. Morphology, disease resistance and yields appeared to be greatly influenced by the wild species GKP 10017 germplasm in plants of the interspecific hybrid population. The potentials of using wild species for improvement of the cultivated peanut are discussed.Paper number 5948 of the journal series of the North Carolina Agricultural Research Service, Raleigh, NC 27650. The investigation was supported in part by ICRISAT and SEA-CR grant no. 701-15-51.     

Interspecific hybridization in the genus Arachis through embryo culture

Summary The embryos of a cultivated tetraploid peanut (Arachis hypogaea), a wild diploid species (A. villosa), and their hybrid embryos, which generally abort in nature, were cultured in vitro and the plants have been successfully transferred to the soil. The hybrids showed triploid chromosome number (3x=30). The significance of wide hybridization in peanut-improvement programs is discussed.     

Rapid estimation of ploidy levels in in vitro-regenerated interspecific Arachis hybrids and fertile triploids

Summary A significant correlation among chromosome number, chloroplast number and pollen grain size was observed using interspecific hybrids (2n=30,60), Arachis hypogaea (2n=40), A. stenosperma (PI 338280) (2n=20), A. batizocoi (PI 468329) (2n=20) and A. villosulicarpa Hoehne (PI 336984) (2n=20), representing four ploidy groups. Both chloroplast number in guard cells and pollen grain size were found to be positively correlated with ploidy, and provided a reliable method to distinguish diploid, triploid, tetraploid and hexaploid plants from each other regardless of their taxonomic backgrounds. These methods in combination with root-tip chromosome counts enabled us to confirm ploidy determinations on all three dissue layers, L₁, L₂ and L₃ (guard cells, microsporocytes and root meristematic cells, respectively), and to detect the chimeric nature of some colchicine-treated tissue culture-derived plants. Screening pollen grains by size and the detection of highly stainable and viable, 30-chromosome pollen grains have enhanced the use of triploid hybrids in peanut breeding programs.     

Crosses between Hordeum vulgare L. and H. bulbosum L. I. Production,morphology and meiosis of hybrids,haploids and dihaploids

Summary From crosses between diploid and autotetraploid cytotypes of Hordeum vulgare L. (cultivated barley) and H. bulbosum L. (bulbous barley grass) diploid, triploid and tetraploid interspecific hybrids were produced. Both directly and after vegetative segregation crosses in either direction also gave rise to haploids and dihaploids resembling H. vulgare. The use of embryo culture was necessary. Plant morphology of the hybrids was much like that of H. bulbosum, although the hybrid plants were less vigorous. Meiosis in the hybrids was more or less disturbed, and this seemed to be the main cause of the high level of sterility.     

Identification of tetraploid regenerants from cotyledons of diploid watermelon cultured in vitro

Summary Adventitious shoots were obtained from the diploid watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai] cultivars Dixielee, Jubilee II, Mickylee, Minilee, and Royal Sweet by culturing excised cotyledons on shoot regeneration medium for six weeks. Tetraploid and diploid regenerants were identified by counting the number of chloroplasts per guard cell pair from leaves of regenerated plants. Cross fertilization of putative tetraploids with diploid pollinators and the production of triploid seed confirmed the efficacy of this approach. The mean number of chloroplasts for tetraploid regenerants was 19.1 whereas diploids averaged 11.2. These values were similar to tetraploid and diploid plants from seed. Ovary diameter, petal, and anther diameter of male flowers, and leaf length by width ratio were also good indicators of plant ploidy. Progeny obtained from self-fertile tetraploids of Mickylee were crossed with various diploid pollinators to produce triploid hybrid seed. All triploid plants from tissue culture-derived tetraploids produced fruit comparable in quality to fruit produced by currently-available triploid hybrids, demonstrating that in vitro tetraploid induction can be used to produce high quality tetraploid plants for use in triploid hybrid seed production.     

Reproductive behaviour and broomrape resistance in interspecific hybrids of sunflower

Interspecific hybrids and backcross generations between the wild perennial species Helianthus resinosus, Helianthus paucifiorus, Helianthus laevigatus, Helianthus nuttallii ssp. nuttallii T. & G. and Helianthus giganteus, resistant to broomrape (Orobanche cernua) and susceptible inbred lines were obtained to study crossability to cultivated sunflower and the transmission and expression of resistance to this parasitic weed. Conventional crosses with all the species tested were successful except for the crosses with diploid H. giganteus, for which embryo rescue techniques were needed to overcome hybrid incompatibility. Pollen viability and seed set were highest for F₁ hybrids with hexaploid species and lowest for those with the diploid H. giganteus. We evaluated F₁, BC₁F₁, some BC₂F₁ plants and the wild and cultivated parents. The wild species and interspecific hybrids were resistant to broomrape infection except for H. nuttallii, which showed segregation, indicating that the resistance is dominant. The crossability and resistance of F₁, and back-cross generations of species with different ploidy levels indicate that the transfer of broomrape resistance to cultivated sunflower is feasible.     

Cytogenetic study of Helianthus laevigatus and its F1 and BC1F1 hybrids with cultivated sunflower, Helianthus annuus

Although the wild sunflower species Helianthus laevigatus has not been extensively studied it may be considered for sunflower breeding as a potential source of desirable genes for Sclerotinia stalk rot resistance and high contents of proteins and linoleic acid in the seed. A set of six H. laevigatus populations was crossed to cultivated sun~ower lines and produced nine F₁ (2-14 plants) and 66 BC₁F₁ hybrid combinations (1-13 plants). Male sterility occurred in F₁ and BC₁F₁ hybrid combinations and pollen viability was lower in the progenies than in the parents (51.6-77.2%in F₁ and in F₁ and 4.8-34.0% in BC₁F₁). Meiosis was normal in the H. laevigatus populations It was found that this tetraploid species also occurred in a hexaploid form Numerous irregularities were observed in the meiosis of the F₁ interspecific hybrids During diakinesis, quadrivalents and hexavalents were recorded in addition to bivalents Dislocated chromosomes and chromosome bridges were present in the other phases The chromosome number in F₁ was 68 (tetraploid). Irregularities in chromosome pairing were observed in the interspecific hybrids at BC₁F₁. There were many univalents, and trivalents quadrivalents and hexavalents were also present The chromosome number in the BC₁F₁ generation ranged from 34 to 60. The occurrence of meiotic irregularities in the F₁ and BC₁F₁ interspecific hybrids indicates that H. laevigatus and the cultivated sunflower differ in genome constitution.     

Variation of chromosome numbers and essential oil components of plants derived from anther culture of the diploid and the tetraploid in Pelargonium roseum

Summary Anthers of the diploid (2n=77) and the colchi-tetraploid (2n=154) Pelargonium roseum were cultured in vitro. In both ploidy level anthers containing uninucleate or binucleate microspores were incubated on a modified White's medium. Calli formed were then subcultured on Murashige and Skoog's medium for organoid differentiation. Plants developed from organoids were transferred to filter paper bridges and after that transplanted into pots. Plants derived from anthers of the tetraploid had diploid chromosome number. Wide variation of their essential oil components suggested their genetic heterogeneity. Further, high correlations between different seasons in the rate of essential oil components showed that the wide variation was due to genetic differences. Therefore, these plants probably originated from pollen grains. On the other hand, plants derived from anthers of the diploid had diploid chromosome number. Small variation and low correlations between different seasons in essential oil components indicated their genetic homogeneity. Their origin was ascribable to the somatic tissues of the mother plant. It is concluded that in plant species in which usual sexual reproduction is difficult, anther culture of chromosome-doubled plants will give a useful method for obtaining genetic variation.     

Introgression of Cajanus platycarpus genome into cultivated pigeonpea, C. cajan

Summary Cajanus platycarpus, an incompatible wild species from the tertiary gene pool of pigeonpea (C. cajan (L.) Millspaugh), has many desirable characteristics for the improvement of cultivated varieties. To necessitate such transfers, embryo rescue techniques were used to obtain F₁ hybrids. The F₁ hybrids were treated with colchicine to obtain tetraploid hybrids, that were selfed to obtain F₂, F₃ and F₄ progenies. All of the hybrids and subsequent progenies had an intermediate morphology between the two parents. Backcrossing of the tetraploid hybrids with cultivated pigeonpea was not possible given embryo abortion, with smaller aborted embryos than those obtained in the F₀ parental cross.As a route of introgression, diploid F₁ hybrids were backcrossed with cultivated pigeonpea and BC₁ progeny obtained by in vitro culture of aborting embryos. BC₂ plants were obtained by normal, mature seed germination. Although embryo rescue techniques had to be used to obtain F₁ and BC₁ plants, it was possible to produce BC₂ and subsequent generations through direct mature seed. Every backcross to cultivated pigeonpea increased pollen fertility and the formation of mature seeds.Special project assistant till December, 2003.