Cytogenetic analysis of two interspecific Citrus allotetraploid somatic hybrids and their diploid fusion parents

Microsporogenesis of two interspecific allotetraploid somatic hybrids, i.e.‘Hamlin’ sweet orange +‘Rough Lemon’ (HR) and ‘Key’ lime +‘Valencia’ sweet orange (KV), was examined and compared with their corresponding diploid fusion parents. Meiotic analysis revealed that only 13.2 and 18.0% of the meiocytes exhibited normal tetrad formation in HR and KV somatic hybrid plants, respectively, and irregular chromosome behaviour with univalent or multivalent pairing occurred. Moreover, meiotic abnormalities such as chromosome bridges, chromosomes orientated away from the equatorial plate, especially lagging chromosomes, which resulted in different sizes of pollen grain, were frequently observed in both somatic hybrids. As expected, the percentage of chromosome abnormalities was much lower in their corresponding diploid fusion parents. HR and KV had 15.4 and 7.2% pollen germinability, respectively, intermediate between their corresponding fusion parents. The meiotic behaviour of these somatic hybrids provided valuable information for their practical utilization in a citrus breeding programme.     

Production and characterization of intergeneric somatic hybrids between Moricandia arvensis and Brassica oleracea

Somatic hybrids were produced between Moricandia arvensis (MaMa, 2n= 28) and Brassica oleracea (CC, 2n= 18) through cell fusion and then characterized by analysing their morphology, cytology, DNA constitution, leaf anatomy and seed fertility. Cell fusion was carried out between greenish protoplasts isolated from the mesophyll of M. arvensis and colourless ones from hypocotyls of B. oleracea. Three plants were generated from one shoot via cuttings and acclimatized in vivo. They closely resembled each other in morphology, exhibiting traits intermediate between the parental species. They were confirmed to be amphidiploids by mitotic and meiotic analyses, being 2n= 46 (MaMaCC), with pollen fertility of about 50%, which was enough to develop the subsequent progenies. Anatomical analysis of the for leaf tissue showed that the bundle sheath cells of the somatic hybrids contained some centripetally arranged organelles, like those of M. arvensis. The hybridity was also confirmed by random amplified polymorphic DNA analysis. Both chloroplast DNA and mitochondrial DNA of the somatic hybrids were estimated to be derived from M. arvensis. In leaf anatomy, the somatic hybrid showed the C₃‐C₄ intermediate trait as in M. arvensis. Many progenies resulted from backcrossing with parental species. The somatic hybrids are expected to be used as bridging plant material to introduce the C₃‐C₄ intermediate trait into Brassica crop species.     

Cytogenetical and molecular investigations on somatic hybrids of Sinapis alba and Brassica juncea and their backcross progeny

Somatic hybrids of Sinapis alba+Brassica juncea (Sal Sal AABB) were synthesized by protoplast electrofusion. They were true genomic allopolyploids since they possessed 60 chromosomes, i.e. the sum of S. alba (2n= 24) and B. juncea (2n= 36) chromosomes. Chromosome pairing was predominantly bivalent with the occasional occurrence of multivalents in the pollen mother cells at diakinesis and metaphase I. Hybrids were completely pollen-sterile, but produced seeds on back-crossing with B. juncea and B. campestris. A total of 37 BC₁ plants were raised from two somatic hybrids (JS-1 and JS-2) and 24 of these were analysed cytologically. The 22 plants originating from the pollinations of somatic hybrids with B. juncea showed a chromosome configuration of 18II+12I and had 42–86% pollen fertility. Two plants from the backcrosses of the somatic hybrid with B. campestris formed 10II +20I, and had 0–4% fertile pollen. Total DNA analysis by probing with pTA71 carrying a full-length 18S–25S rDNA fragment of the wheat nuclear genome revealed that the two somatic hybrids possessed all the characteristic bands of both the species, confirming their hybridity. Probing with the mitochondrial coxI and atp9 genes indicated mitochondrial genome recombination in the hybrids. Hybridization with chloroplast-specific psbD indicated that both the somatic hybrids possessed the cp genome of S. alba origin.     

Production of somatic hybrid plants between Iris ensata Thunb. and I. germanica

Wide hybridization that cannot be attained through conventional sexual crosses, can now be approached by somatic hybridization. Protoplasts of I. ensata and I. germanica were fused by electrofusion. For the selection of somatic hybrids, protoplasts of I. ensata which did not form colonies in protoplast culture and protoplasts of I. germanica which had regeneration ability for only albino shoot were used in symmetric fusion. On the other hand, the protoplasts of I. ensata and I. germanica protoplasts which were inactivated by iodoacetamide (IOA) treatment were used in asymmetric fusion. Five-six months after cell fusion, green plants were obtained in the symmetric and asymmetric fusion. In the random amplified polymorphic DNA (RAPD) analysis, the green plants had bands specific to both parental species. Therefore, these plants were somatic hybrids between I. ensata and I. germanica. This revised version was published online in July 2006 with corrections to the Cover Date.     

Production of somatic hybrids between S. tuberosum L. and late blight resistant Mexican wild potato species

Interspecific somatic hybrids between dihaploid breeding clones of potato, S. tuberosum and two accessions of wild Mexican species S. pinnatisectum and the hybrid line S. pinnatisectum × S. bulbocastanum were regenerated following electrofusion of mesophyll protoplasts to combine important agricultural traits of S. tuberosum and a high level of late blight resistance from selected wild accessions. In two fusion combinations 239 calli were regenerated; 162 from 195 calli analysed were identified as hybrids by means of isozyme analysis of peroxidases and, for some hybrid clones, by RAPD analysis. Depending on the fusion combination, 47–89 percent of the somatic hybrids had the expected ploidy level and 7–16 percent were mixoploids. Somatic hybrids were phenotypically intermediate as compared to their parents and some of them were able to be backcrossed sexually with potato. Fertility and crossability depended on combination and ploidy level of the somatic hybrids. In tests with detached leaves the wild partner clones had a high late blight resistance score of 8,6 and 8,9; the susceptible tuberosum-partners of 2,8 and 3,5, respectively. Nearly 25 percent of somatic hybrids had a resistance level of 6 or higher in the first year of assessment. The average resistance value of most somatic hybrids was lower than the average parental level. The reasons for variation in resistance values are discussed in connection with the practical application of fusion hybrids. This revised version was published online in July 2006 with corrections to the Cover Date.     

Regeneration and characterization of somatic hybrid plants of Citrus sinensis (sweet orange) and Citrus micrantha, a progenitor species of lime

Most commercial citrus fruit species and cultivars are inter- and intra-specific hybrids. Conventional hybridization in citrus is largely handicapped by apomixis and long juvenility. As an alternative, somatic hybridization via protoplast fusion has been employed to create novel citrus germplasm. Witches' broom disease of lime (WBDL) emerged in Oman during the 1970s, which has been spreading to the neighboring countries. The disease is extremely destructive. A possible solution to the WBDL problem is to develop resistant hybrids. Resistance is available among the citrus relatives of lime i.e., sweet orange. Somatic hybrids combining sweet orange with lime have been produced but the fruit characteristics are different from lime. Herein, we report the development of somatic hybrids between a progenitor of lime (Citrus micrantha) and sweet orange (Citrus sinensis) in efforts to recreate a lime-like fruit using sweet orange as a donor of disease resistance gene(s). Successful somatic hybridization was verified by ploidy analyses using flow cytometry and RAPD analyses. This is the first report of using a progenitor species in somatic hybridization experiments in efforts to resynthesize an improved lime.     

Production of intergeneric hybrids between Raphanm and Moricandia

Intergeneric F₁ hybrids between Raphanus sativus (2n = 18, RR) and Moricandia arvensis (2n = 28, MaMa) have been produced through ovary culture followed by embryo culture, when M. arvensis was used as a pistillate parent. Six BC₁ plants were also obtained through ovary culture followed by embryo culture in the backcross of an amphidiploid F₁, hybrid with R. sativus cv. 'Pink ball'. Two BC₁ plants were ses-quidiploids (2n = 32, MaRR), and the other BC₁, plants were hyperploid with 2n = 55, having MaMaRRR genomes. BC₂, seeds were obtained by conventional pollination in the successive backcross of two sesquidiploid BC₁, plants with R. sativus cv. 'Pink ball'. Their seed set percentages were 12.7% and 17.0%, respectively. These novel hybrid plants and derived progenies may be valuable materials for the genetic investigation and breeding of Brassiceae , including R. sativus.     

Wide somatic hybrids of Citrus with its related genera and their potential in genetic improvement

Citrus wild relatives are an untapped germplasm reservoir, which possesses many elite resistance traits. Genetic introgression into Citrus by conventional methods has been greatly hindered by polyembryony, pollen / ovule sterility, sexual / graft incompatibility, long juvenility etc. Somatic hybridization via protoplast fusion may make it possible to transfer genes from wild relatives to Citrus. To date, more than sixty sexually compatible or incompatible intergeneric somatic hybrids between Citrus and its various related wild genera have been produced by PEG - orelectrically - induced fusion. These wide somatic hybrids were identified by morphology, cytology, isozyme, RAPD and RFLP analyses. Genetic variation or recombination was also revealed in some of them. Several sexually compatible combinations have flowered and set fruits. The agronomic performance of these wide somatic hybrids is diverse, and the current results are not conclusive. Somatic hybrids are being tested as rootstocks. The prospects of wide somatic hybridization of Citrus with its wild relatives are discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Production and characterization of intergeneric hybrids between Brassica oleracea and a wild relative Moricandia arvensis

Intergeneric crosses were made between Brassica oleracea and Moricandia arvensis utilizing embryo rescue. Six F₁ hybrid plants were generated in the cross‐combination of B. oleracea × M. arvensis from 64 pods by the placenta‐embryo culture technique, whereas three plants were produced in the reciprocal cross from 40 pods by the ovary culture technique. The hybrid plants were ascertained to be amphihaploid with 2n = 23 chromosomes in mitosis and a meiotic chromosome association of (0–3)II + (17–23)I at metaphase I (M I). In the backcross with B. oleracea, some of these hybrids developed sesquidiploid BC₁ plants with 2n = 32 chromosomes that predominantly exhibited a meiotic configuration of (9II + 14I) in pollen mother cells. The following backcross of BC₂ plants to B. oleracea generated 48 BC₃ progeny with somatic chromosomes from 2n = 19 to 2n = 41. The 2n = 19 plants showed a chromosomal association type of (9II + 1I) and a chromosomal distribution type of (9¹/₂ + 9¹/₂) or (9 + 10) at M I and M II, respectively. These facts might suggest that they were monosomic addition lines (MALs) of B. oleracea carrying a single chromosome of M. arvensis that could offer potential for future genetic and breeding research, together with other novel hybrid progeny developed in this intergeneric hybridization.     

Isolation of two microsatellite markers from BAC clones of the Vf scab resistance region and molecular characterization of scab‐resistant accessions in Malus germplasm*

A polymerase chain reaction (PCR)‐based method was developed to isolate microsatellite markers from large‐insert genomic DNA clones of bacterial artificial chromosome (BAC) libraries. The method is fast and economic since it does not require subcloning. It was applied to isolate a microsatellite marker from a BAC clone of the chromosomal region containing the apple scab resistance gene Vf. The Vf gene of Malus floribunda 821 is the most widely used source of scab resistance in apple breeding. A second microsatellite was found on the extremity of a BAC clone flanking the Vf locus. The two microsatellites allowed the identification of the presence of the Vf gene in the scab‐resistant accessions M. micromalus SA573‐3, ‘Golden Gem’, M. prunifolia 19651 and MA 16 not previously known to carry this gene. They were also used to verify the correctness of the published genealogical tree of the Vf cultivar ‘Florina’, in which a probable mistake was identified. This analysis shows the importance of genotyping the Vf locus when choosing scab‐resistant germplasm as parents in breeding programmes.     

Production of intergeneric hybrids between Brassica and Sinapis species by means of embryo rescue techniques

Reciprocal crosses were made to produce intergeneric hybrids among seven species of Brassica and three species of Sinapis with the aid of embryo rescue techniques. Ovule culture showed better responses in terms of hybrid plant development. Altogether, hybrids from 8 different combinations were obtained from the crosses between B. campestris var. trilocularis × S. turgida, B. campestris var. pekinensis × S. arvensis, S. arvensis × B. campestris var. pekinensis, B. oleracea var. alboglabra × S. alba, B. oleracea var. alboglabra × S. turgida, B. carinata × S. alba, B. carinata × S. arvensis, and B. carinata × S. turgida. Reciprocal crosses yielded no hybrid except in the combination of S. arvensis × B. campestris var. pekinensis. Among them hybrids from 6 combinations were established in the glass house. The hybridity of the plants was confirmed by morphological characters, pollen stainability, chromosome number and by isozyme analysis. Hybrids of four combinations out of six turned out as true hybrids, one as sesquidiploids and the another one as false hybrid plant. This revised version was published online in August 2006 with corrections to the Cover Date.     

Development of chloroplast‐specific microsatellite markers for molecular characterization of alloplasmic lines and phylogenetic analysis in wheat

Wheat (Triticum aestivum L.) is strictly a self‐pollinated crop, where hybrid breeding requires well‐characterized cytoplasmic male sterile (CMS) lines. The CMS has mostly been developed by substituting nuclear genome of wheat into the cytoplasm from wild relatives. Molecular characterization of 90 genotypes including 82 CMS lines originating from five different species, namely Aegilops speltoides, Ae. kotschyi, Ae. variabilis, Triticum araraticum and T. timopheevii, and eight popular varieties was carried out. Consequently, a set of 25 microsatellite markers specific to chloroplast (cpSSRs) were designed and successfully validated for specificity of amplification. A total of 15 cpSSRs (60%) were found polymorphic, of which three cpSSRs (TaCM7, TaCM8 and TaCM11) in genic region and twelve cpSSRs were located in intergenic region. Phylogenetic analysis of genotypes using cpSSRs revealed two major groups well in accordance with respective origin. A set of cpSSRs and phylogeny of CMS belonging to different origins developed, which will be helpful for the improvement in CMS system in wheat. The genic cpSSRs can be used for the allele mining and evolutionary studies.     

Development of interspecific somatic hybrid cell lines in cultivated jute and their early characterization using jute chloroplast RFLP marker

A strong sexual incompatibility barrier that exists between the two cultivated jute species, Corchorus capsularis and Corchorus olitorius, limits the scope for improvement through genetic introgression. Protoplast fusion was carried out to generate interspecific hybrid cell lines. Cotyledonary cell protoplasts of C. capsularis and anthocyaninpigmented hypocotyl protoplasts of C. olitorius were used in the fusion experiments, which appeared to be visually useful in the early selection of the fused products. A chloroplast DNA (cpDNA) marker was developed in jute, which showed species‐specific hybridization patterns with EcoRI‐digested total genomic DNA of C. capsularis and C. olitorius. This cpDNA marker was used in the characterization of the somatic hybrid cell lines at their early stages of growth. Evidence for the presence of both types of cpDNA in the hybrid cell lines was obtained when the total genomic DNA of 4‐ to 7‐month‐old hybrid cell lines was challenged with the chloroplast DNA marker through Southern analysis. It was shown that the early segregation of the parental chloroplasts did not occur in jute, although this is common in other plant species. The hybrid nature of the fused cell lines could also be identified through peroxidase isozyme analysis. Isozyme banding patterns were complex and varied among the hybrid cell lines.     

Production and cytogenetics of intergeneric hybrids between Triticum durum-Dasypyrum villosum amphidiploid and Psathyrostachys huashanica

Summary Intergeneric crosses between Triticum durum-Dasypyrum villosum (2n=42, AABBVV), and Psathyrostachys huashanica (2n=14, N ^h N ^h ) were made, the seed set was 1.67%. Intergeneric hybrid were successfully obtained by means of embryo culture for first time. The average chromosome pairing in the hybrid (ABVN ^h ) was 26.61% univalents, and 0.69 bivalents. The chiasmata per cell was 0.69. The chiasmata was higher than that in Triticum durum dihaploid (AB), and lower than that in T. durum-Dasypyrum villosum trihaploid (ABV). The result indicated that the N ^h genome of Psathyrostachys huashanica has no homology with the V genome of Dasypyrum villosum, and the A and B genomes of Triticum durum. The coenocytism, micronuclei cell and variation in chromosome numbers were also observed. The F₁ hybrid was crossed with Triticum aestivum (AABBDD), and resulted in seed set. The hybrid of T. durum-D. villosum amphidiploid x P. huashanica showed partial fertility. It made the possibility for chromosome manipulation among Triticum aestivum, Dasypyrum villosum and Psathyrostachys huashanica.     

Production and characterization of intergeneric diploid cybrids derived from symmetric fusion between Microcitrus papuana Swingle and sour orange (Citrus aurantium)

Plants were regenerated from intergeneric somatic hybridization between embryogenic protoplasts of Microcitrus papuana Swingle and leaf-derived protoplasts of sour orange (Citrus aurantium L.) via electrofusion. The regenerated plants were morphologically similar to the leaf parent in growth vigor, leaf and branch structure. FCM analysis showed that they were diploids. Simple-sequence-repeat (SSR) and cleaved-amplified-polymorphic-sequence(CAPS) were employed for hybridity characterization. SSR banding patterns of the regenerated plants were identical to the leaf parent, sour orange, indicating that they possessed nuclear component derived from sour orange. DNA amplification with chloroplast and mitochondrial universal primers, followed by restriction endonuclease digestion, revealed polymorphism between the fusion parents. Therefore, this method was used to determine the cytoplasmic compositions of the regenerated plants. Banding patterns for all the polymorphic primer/enzyme combinations of the regenerated plants were similar to those of the embryogenic parent, M. papuana, suggesting that only the cytoplasmic components derived from the embryogenic parent were present in the regenerated plants. FCM, SSR and CAPS demonstrated that intergeneric diploid cybrids have been successfully obtained by symmetric fusion. Related results concerning nuclear and cytoplasmic composition of previous diploid somatic hybrids and potential mechanism for regeneration of such kind of plants are discussed herein. This revised version was published online in July 2006 with corrections to the Cover Date.     

Successful backcrosses of somatic hybrids between Sinapis alba and Brassica oleracea with the Brassica oleracea parent

Somatic hybrids between Sinapis alba (2n= 24) and Brassica oleracea (2n= 18) have been backcrossed with the B. oleracea parent. Whereas backcrosses with the diploid B. oleracea parent were unsuccessful, 344 BC₁ seeds could be obtained from inter-valence crosses with tetraploid B. oleracea (2n= 4x= 36). The investigated 96 BC₁ plants segregated for morphological traits and for fertility. They were backcrossed with diploid B. oleracea or self-pollinated, depending on their male fertility. The BC₁F₂ and BC₂ progenies segregated well for the morphological traits. Disturbances were observed especially in the generative phase (flower development and pollen fertility). Both male fertile and male sterile BC₁F₂ and BC₂ plants were obtained and backcrossed or self-pollinated with the B. oleracea parent. The presence of either one of the parental or the cybrid organelle genomes was detected. In the progenies, a stable maternal inheritance of the organelle genome patterns was observed. Isozyme analyses revealed polymorphism for the leucine aminopeptidase (LAP) which was used for the identification of S. alba genes in the progenies. Cytological investigations showed a clear differentiation between the BC₁F₂ and BC₂ plants. Whereas the BC₁F₂ plants possess large numbers of chromosomes ranging from 34 to 40, the BC₂ material was strongly reduced to chromosome numbers ranging from 20 to 22. Preliminary investigation of the meiosis suggests the possibility of introgressions of S. alba-DNA into the B. oleracea genome.