Production and molecular characterization of Citrus intergeneric somatic hybrids between red tangerine and citrange

Somatic hybridization has been an effective and successful technique for plant improvement. In this paper, embryogenic callus protoplasts of red tangerine (Citrus reticulata Blanco) were electrically fused with mesophyll protoplasts from citrange (C. sinensis × P. trifoliata, a Chinese local strain) in an effort to produce complementary tetraploid citrus rootstocks. Regenerated embryoids grew slowly and were vulnerable to browning. Twelve plants were finally regenerated, rooted and transplanted into a greenhouse. Root‐tip chromosome counting of five randomly‐selected plants revealed most cells were tetraploid (2n = 4x = 36), but aneuploid cells also existed. Flow cytometry analysis further confirmed their tetraploid nature. Nuclear simple sequence repeat (SSR) analysis verified their hybridity. Further mitochondrial genome analysis by restriction fragment length polymorphism and cleaved amplified polymorphic sequence revealed their mtDNA banding pattern was identical to that of red tangerine, the embryogenic callus parent; while their chloroplast DNA inheritance was random as revealed by chloroplast SSR analysis, in addition to cpDNA co‐existence detected in one plant. Cytological and molecular analysis indicated that somatic hybrid plants between red tangerine and citrange had been successfully obtained.     

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.     

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.     

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.     

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.     

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 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.     

A new source of resistance against coffee leaf rust from New-Caledonian natural interspecific hybrids between Coffea arabica and Coffea canephora

The development of cultivars resistant to coffee leaf rust caused by the fungal pathogen Hemileia vastatrix is a priority in coffee breeding. However, only very few descendants of interspecific hybrids between Coffea arabica and diploid relative species have been used as resistance source. Identification of new sources of resistance appeared therefore particularly worthwhile. Hybrid plants derived from interspecific hybridization between C. arabica and Coffea canephora and found in neo‐natural coffee tree populations of New Caledonia were therefore investigated. Amplified Fragment Length Polymorphism and microsatellites amplification were used to evaluate the genetic diversity of 14 hybrid plants, and rust resistance was evaluated by inoculation with a panel of rust races representing a large variability in virulence. An important genetic diversity was characterized in hybrid plants originating from introgressions into C. arabica from various C. canephora progenitors. On the 14 plants tested for leaf rust resistance, eight appeared resistant to all races investigated. Such plant material should represent a highly valuable resource for C. arabica breeding against coffee leaf rust.     

The generation of novel species hybrids between garden dahlias and Dahlia macdougallii to increase the gene pool for variety breeding

To transfer new traits into the gene pool of garden dahlias (Dahlia variabilis), crosses between garden dahlias (2n = 64) and the epiphytic species Dahlia macdougallii (2n = 32) from the section Epiphytum were conducted. Six hybrid plants were obtained. The hybrid status was verified using three SSR markers. In addition, flow cytometry was performed to determine the genome size of the hybrids and to show that the hybrids were hexaploid as expected from crosses between tetraploids and octoploids. The open pollinated progeny of the hybrids produced four progeny with octoploid genomes. The hybrids exhibited indeterminate vegetative growth and the formation of flowers from axillary buds, similar to the father D. macdougallii. This result is of interest for breeding new varieties of dahlia with traits that are not present in the current gene pool.     

Characterization of morphological variation and cold resistance in interspecific somatic hybrids between potato (Solanum tuberosum L.) and S. brevidens Phil.

Summary Somatic hybrids between Solanum tuberosum L. cv. Gracia (2n=4x=48) and Solanum brevidens Phil. (2n=2x=24) were produced via fusion of mesophyll protoplasts. Selection of the protoplast derived putative hybrid calli was based on their vigorous growth. Additive isozyme patterns and chromosome numbers as well as the expression of parental morphological characters have proved the hybrid origin of the selected regenerants. Extensive chromosome loss during the regeneration process resulted in aneuploid hybrids with high frequency. Genomic instability could not be detected in these plants during the period of vegetative propagation. Regenerants from hybrid tissues exhibited wide morphological variation especially in tuber formation. The detailed morphological analysis based on the use of multivariate method (principal component analysis, PCA) enabled to identify morphological groups among the hybrid clones. The positioning of hybrid clones in the PCA space could not be correlated with chromosome numbers. The genomic ratio represented by the tetraploid and diploid parents influenced the morphology of somatic hybrid population according to the applied analytical system. Two selected hybrid clones have exhibited an intermediate degree of frost tolerance compared to the parents, based on the recovery of plants from lower buds after freezing of potted plants.     

Chromosome and cytoplasm analyses of somatic hybrids between onion (Allium cepa L.) and garlic (A. sativum L.)

Chromosomes and cytoplasms were analyzed in two lines of a somatic hybrid between onion (Allium cepa L.) and garlic (A. sativum L.). One line of the somatic hybrid had 40 chromosomes and the other 41chromosomes. Genomic in situhybridization successfully revealed the chromosome constitution of the two lines. One line had 20 chromosomes from onion and17 chromosomes from garlic, and the other had 21 chromosomes from onion and 17chromosomes from garlic. Interestingly, both lines had three chimeric chromosomes. PCR-RFLP analyses of chloroplast and mitochondrial DNAs of both lines showed that these were identical to the onion parent. This revised version was published online in July 2006 with corrections to the Cover Date.