Production Of Somatic Hybrids Research Articles

Utilization of somatic fusion techniques for the development of HLB tolerant breeding resources employing the Australian finger lime (Citrus australasica).

The Australian finger lime is a unique citrus species that has gained importance due to its unique fruit characteristics and perceived tolerance to Huanglongbing (HLB), an often-fatal disease of citrus trees. In this study, we developed allotetraploid finger lime hybrids and cybrids by utilizing somatic cell fusion techniques to fuse diploid ‘OLL8’ sweet orange or ‘Page’ tangelo callus-derived protoplasts with finger lime (FL) mesophyll-derived protoplasts. Six somatic fusions were regenerated from the ‘OLL8’ + FL fusion, while three putative cybrids were regenerated from the ‘Page’ + FL fusion. Ploidy levels and nuclear-expressed sequence tag derived simple sequence repeat (EST-SSR) markers confirmed the somatic hybrid production, and mitochondrial DNA primer sets confirmed the cybrid nature. Several trees produced by the somatic fusion remained HLB negative even after 6 years of growth in an HLB-endemic environment. Pathogenesis related (PR) and other genes that are often upregulated in HLB-tolerant trees were also upregulated in our somatic fusions. These newly developed somatic fusions and cybrids could potentially be used as breeding parents to develop the next generation of improved HLB-tolerant rootstocks and scions.

Characteristics of fertile somatic hybrids of G. hirsutum L. and G. trilobum generated via protoplast fusion

Fertile somatic hybrids between tetraploid upland cotton G. hirsutum L. cv. Coker 312 and wild cotton G. trilobum were generated by symmetric electrofusion. Comparisons of morphology, combined with flow cytometric, RAPD, SRAP and AFLP analyses confirmed the hybrid nature of the regenerated plants. The hybrids differed morphologically from the parent plants. Flow cytometric analysis showed that the hybrids had DNA similar in amount to the total combined DNA content of the two parents, and the use of molecular markers revealed that the hybrids contained genomic fragments from both fusion parents, further indicating the hybrid nature of the regenerated plants. The stability of the morphological features of the hybrids was examined in following generations. The hexaploid fusion plants showed strong photosynthesis and a high expression level of some photosystem-related genes. Our results suggest that novel traits may be incorporated in cotton breeding programs through the production of somatic hybrids and the backcrossing of these plants with elite cultivars.

Myo-inositol increases the plating efficiency of protoplast derived from cotyledon of cabbage (Brassica oleracea var. capitata)

This study describes the effect of myo-inositol on sustained cell division and plant regeneration from cotyledon-derived protoplast of cabbage (Brassica oleracea var. capitata). Freshly isolated protoplasts were cultured in modified Murashige and Skoog (MS) medium removed ammonia ions and containing <TEX>$0.4\;mg\;l^{-1}$</TEX> thiamine HCl, <TEX>$100\;m</TEX><TEX>g\;l^{-1}$</TEX> myo-inositol, <TEX>$2\;mgl^{-1}$</TEX> 2,4-D, <TEX>$0.5\;mgl^{-1}$</TEX> BA, <TEX>$30\;gl^{-1}$</TEX> sucrose and several concentrations of myo-inositol (2, 4, 6, 8, 10% (w/v)) as an osmotic stabilizer. After 3 weeks of culture in the dark at <TEX>$25^{\circ}C$</TEX>, the plating efficiency of cabbage protoplasts reached to <TEX>$22.5{\pm}2.9%$</TEX> when cultured in modified MS medium supplemented with <TEX>$2\;mgl^{-1}$</TEX> 2,4-D, <TEX>$0.5\;mgl^{-1}$</TEX> BA, <TEX>$30\;gl^{-1}$</TEX> sucrose and 8% (w/v) of myo-inositol at a density of <TEX>$2{\times}10^5$</TEX> protoplasts/ml. Rapidly growing cell colonies after 3 weeks of culture were transferred to the same culture medium removed osmoticum. To induce shoot regeneration from calluses, calluses with about 2 mm in diameter were transferred to the MS medium containing <TEX>$2\;mgl^{-1}$</TEX> BA and <TEX>$0.5\;mgl^{-1}$</TEX> NAA. After further three weeks of incubation onto the medium in the light, green shoots were formed on the surface of calluses at a frequency of 30%. Upon transfer to half-strength MS basal medium, roots were formed onto the bottom of regenerated shoots without auxin treatments. These regenerated plantlets were successfully acclimatized to soil transfer, grown to normal mature plants. The cabbage protoplast culture system established in this study could be applied for production of somatic hybrids or cybrids by asymmetric protoplast fusion and mass proliferation of elite somatic clones of cabbage.

Somatic hybridization for citrus rootstock breeding: an effective tool to solve some important issues of the Mediterranean citrus industry

The prevalence of sour orange rootstock in the southern and eastern part of the Mediterranean Basin is presently threatened by the spread of Citrus Tristeza Virus (CTV) and its main vector Toxoptera citricida, combined with abiotic constraints such as drought, salinity and alkalinity. The search for alternative CTV-resistant rootstocks that also withstand the other constraints is now considered an urgent priority for a sustainable citrus industry in the area. Complementary progenitors can be found in citrus germplasm to combine the desired traits, particularly between Poncirus and Citrus genera. The production of somatic hybrids allows cumulating all dominant traits irrespective of their heterozygosity level, and would appear to be an effective way to solve the rootstock challenge facing the Mediterranean citrus industry. This paper presents the results obtained during a regional collaborative effort between five countries, to develop new rootstocks by somatic hybridization. New embryogenic callus lines to be used for somatic hybridization have been created. Protoplast fusions have been performed at CIRAD and IVIA laboratories, focusing on intergeneric combinations. Analysis of ploidy level by flow cytometry and molecular markers confirmed the acquisition of new interesting tetraploid somatic hybrids for six combinations. Diploid cybrids with intergeneric (Citrus×Poncirus) nucleus and C. reticulata or C. aurantifolia mitochondria were also identified for four combinations. The agronomical performance of a pre-existing somatic hybrid between Poncirus trifoliata and Citrus reticulata was validated in calcareous soils in Morocco. Somatic hybridization is now integrated into the breeding programs of the five Mediterranean countries.

Production of somatic hybrids between satsuma mandarin (Citrus unshiu) and navel orange (Citrus sinensis) by protoplast fusion

We have regenerated altotetraploid plants that are interspecific somatic hybrids between Citrus sinensis Osbeck cv. Yoshida navel orange and Citrus unshiu Marc cv. Okitsu satsuma mandarin. Protoplasts isolated from ‘Yoshida’ leaves were chemically fused with call us-derived protoplasts from ‘Okitsu’. After 6 months of culture, 102 plants were obtained. These hybrids were identified by differential leaf morphology, DNA fluorescence intensity, and DNA analysis. Ploidy analysis via the flow cytometry revealed that 15 of the 102 plants were tetraploids, with the rest being diploids that morphologically resembled their mesophyll parent. SRAP analysis confirmed that 9 of the tetraploid plants were allotetraploid somatic hybrids. These will be utilized as a possible pollen parents for improving seedy citrus cultivars, e.g., ponkan, mandarin, lemon and kumquat, in order to produce triploid seedless hybrids.

Regeneration of plants from Ipomoea cairica L. protoplasts and production of somatic hybrids between I. cairica L. and sweetpotato, I. batatas (L.) Lam.

Plants were regenerated from mesophyll protoplasts of Ipomoea cairica L., a wild relative of sweetpotato (Ipomoea batatas (L.) Lam.), and somatic hybrids between I. cairica L. and sweetpotato cv. Xushu 18 were obtained by PEG-mediated method. I. cairica L. protoplasts were isolated from the leaves of in vitro grown plants and cultured in a modified MS medium containing 0.05 mg l−1 2,4-D and 0.5 mg l−1 kinetin. Nine weeks after plating, the obtained small calluses up to about 2 mm in diameter were transferred to solid MS medium supplemented with 0.05 mg l−1 2,4-D and 0.5 mg l−1 kinetin for callus proliferation. Three weeks after transfer, the calluses were transferred to MS medium supplemented with 0–1.0 mg l−1 IAA and 1.0–3.0 mg l−1 BAP and further to hormone-free MS medium for plant regeneration. The frequencies of calluses forming plants ranged from 6.0% to 41.3% based on the different concentrations of IAA and BAP, and 2.0 mg l−1 BAP gave the highest regeneration frequency of protoplast-derived calluses in I. cairica L.. The regenerated plants, when transferred to soil, showed 100% survival. No morphological variations were observed. Mesophyll protoplasts of I. cairica L. were fused with protoplasts isolated from embryogenic suspension cultures of Xushu 18 by PEG-mediated method. The fused products were cultured with the best protoplast culture system of I. cairica L.. Finally, 114 plants were produced from 63 of the 182 calluses derived from the fused protoplasts, and 46 plants of them were confirmed to be somatic hybrids through peroxidase isozyme, RAPD, morphological and cytological analyses.

An Improved Protocol for Microcallus Production and Whole Plant Regeneration from Recalcitrant Banana Protoplasts (Musa spp.)

One important limitation for routine production of somatic hybrids in banana (Musa spp.) is the difficulty in protoplast regeneration. To facilitate protoplast regeneration in banana, the crucial step of microcallus production was optimised for the following parameters: nurse culture medium, duration of microcalli on nurse culture, differing nurse cells, and filter composition. A comparative study between two nurse cell media, Ma2 and PCM, significantly affected the number of microcalli produced, which was 90 × 103 per Petri dish on Ma2 with 0.5 μM zeatin and 9.0 μM 2,4 D, and 30 × 103 per Petri dish on PCM. Moreover, continuous production of microcalli was achieved on Ma2 and the frequency of embryogenic cell aggregates was higher among microcalli on Ma2-medium. However, no cell division was observed in protoplasts cultured on Ma2 in which nurse cells were maintained for 2 weeks suggesting a requirement of effective presence of nurse cells for cell division of banana protoplasts. Use of a filter in conjugation with nurse cells resulted in greater than 7-fold increase in the number of microcalli. Flow cytometry analysis of 124 protoplast-derived plants showed the presence of hexaploid plants (mother plant is triploid) at the frequency of 4%. Together, these data are indicative of the complex factors involved in the regulation of plant cell division and growth. Each individual aspect must be optimised for efficient protocol development.

Cell lines and plants obtained after protoplast fusions of Rosa+Rosa, Rosa+Prunus and Rosa+Rubus

Protoplasts of three Rosa cultivars were fused with each other, with protoplasts of Prunus `Colt' and with protoplasts of Rubus laciniatus, using polyethylene glycol 4000 as a fusogen. Protoplasts of Prunus were incapable of cell division and those of Rosa and Rubus were disabled by treatments with metabolic inhibitors, either iodoacetate (IOA) or rhodamine 6-G (R6G). Parental protoplasts were then fused in combinations that required complementation for their survival. RAPD analysis of 41 fusion-derived cell lines showed that two lines resulting from fusions of Rosa + Rosa and one from a fusion of Rosa + Prunus, contained some DNA markers from both fusion partners. The others contained markers of only one fusion parent. This showed that after protoplast fusion, the heterokaryons did not develop into cell lines with stable hybrid nuclei. Plants regenerated from cell lines derived from Rosa + Prunus and Rosa + Rubus fusions contained DNA markers of only Rosa and their DNA amounts were no greater than that of the Rosa parent. However, they differed morphologically from the Rosa parent to a remarkable degree, possibly because they inherited undetected genes of Prunus or Rubus, or because they were somaclonal variants of the Rosa parent. Alternative strategies for the production of somatic hybrids are discussed.

Production of somatic hybrids between Brassica oleracea and the C3–C4 intermediate species Moricandia nitens

Protoplast fusion between Brassica oleracea and Moricandia nitens, a C3–C4 intermediate wild species, was carried out. Four hundred and twenty five plants were regenerated from 1995 calli. More than 90% of the regenerated plants were verified as true intergeneric hybrids on the basis of morphological observation and molecular-marker analysis. The hybrids were morphologically intermediate between both fusion parents. Variations in flower color and petal number were also observed. The chromosome number and pollen fertility varied across the individual hybrids. Although after self-pollination pollen germinated on the stigma and pollen tubes were visible in the style, the pods did not develop properly without in vitro culture. Measurements of the CO2 compensation point revealed that six out of eight hybrid plants expressed a gas-exchange character that was intermediate between the C3–C4M. nitens and C3B. oleracea parents.

Production of somatic hybrids between Diospyros glandulosa and D. kaki by protoplast fusion

Interspecific somatic hybrids between Diospyros glandulosa (2n=2x=30) and D. kaki cv. Jiro (2n=6x=90) were produced by electrofusion of protoplasts. Protoplasts were isolated from calli derived from leaf primordia, fused electrically, and cultured by agarose-bead culture using a modified KM8p medium. Flow cytometry revealed that the nuclear DNA content was the sum of those of D. glandulosa and D. kaki cv. Jiro in 149 of the 166 calli obtained. RAPD analysis showed that the 149 callus lines yielded specific bands for both D. glandulosa and D. kaki cv. Jiro and further confirmed that they were interspecific somatic hybrid calluses. Shoots were regenerated from 63 of the 149 interspecific hybrid calluses. Chloroplast DNA analysis by PCR-RFLP, flow cytometric determination of nuclear DNA content, and RAPD analysis revealed that the 63 interspecific hybrid shoot lines contained the nuclear genomes from both parents but only the chloroplast genome from D. glandulosa. Microscopic observation of root tip cells demonstrated that somatic chromosome number of the interspecific hybrids was 2n=8x=120.

Genomic stability and stability of expression in genetically modified plants

This review provides a survey of those factors that might influence genetic stability of genetically modified organisms and somatic hybrids in breeding programmes. In this respect several aspects may be distinguished: (i) host genomic factors that might influence genetic stability, (ii) events related to the introduction of new DNA into the genome and their effect on genetic stability, (iii) stability of gene expression of newly introduced DNA, and (iv) stability of the modified genome. In our view a gene is defined as being stable if it inherits according to Mendelian laws. Obviously, this can be valid only for nuclear genes. Non-Mendelian inheritance may be caused by intrinsic genomic factors or be the result of skewed segregation during meiosis. Newly introduced DNA may be stably integrated into the genome, yet data on its site of integration is limited. The level of expression and, thus, the strength of the related trait, may vary. Variation in expression may depend on the construct, such as the promoter or additional sequences such as MAR elements or the coding sequence itself, the site of integration and the species used. Another, and undesired, phenomenon is the silencing of expression of introduced genes. The kinds of silencing described depend on the relative position in the genome of the genes involved, cis vs. trans and whether only one or all genes are silenced. Instability of expression generally becomes visible within a few generations, but once expression is stable it is supposed to remain so provided the environment does not change dramatically. Although the production of somatic hybrids seems to be a promising technique to obtain new genetic material and even though numerous hybrids have been made, only a few follow-up studies have been published. Therefore the use of somatic hybrids in breeding programmes is limited.

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.

Production of Somatic Hybrids and Cybrids in the Rutaceae Family and Application to Citrus Breeding.

Production of Somatic Hybrids and Cybrids in the Rutaceae Family and Application to Citrus Breeding.

Solanum tuberosumとSolanum morelliformeとの体細胞雑種の作出

Solanum tuberosumとSolanum morelliformeとの体細胞雑種の作出

Production of Somatic Hybrids between Tomato (Lycopersicon esculentum) and Night Shade (Solanum lycopersicoides) by Electrofusion

Mesophyll protoplasts of tomato (Lycopersicon esculentum Mill. cv. 'Ohgata Zuiko') and Solanum lycopersicoides were fused by electrofusion method to develop fertile tetraploid somatic hybrids. Following electrofusion the protoplasts were plated in modified TM2 (MTM2) medium in which S. Iycopersicoides protoplast could not form callus. The putative hybrid calli were regenerated on MS3ZG regeneration medium in which tomato protoplasts derived calli could not regenerate shoots. Tomato protoplasts were partially inactivated with iodoacetamide (IOA) for the easy selection of heterokaryons. Three hundred regenerated plants were grovvn to maturity in a green house. The hybridity of the plants was argued by morphological, isozyme and random amplified polymorphic DNA (RAPD) analyses. All plants were heterotic in their growth, expressing an erect stem with a prominent internode like tomato, as well as days to flowering, perennial habit, free anther without sterile tip like S. Iycopersicoides. Several traits, such as leaf margin, inflorescence, flower and fruit color were intermediate between both parents. The electrophoretic pattern of PGI (.phosphoglucoisomerase), GOT (.glutamate oxaloacetate transaminase) and PG[M (phosphoglucomutase) confirmed the hybrid nature of the nuclei of all the tested plants. RAPD markers generated from 35 randomly selected plants, using eight decamer primers reconfirmed the hybrid nature of their nuclear genomes. Cyt.ological study of 20 randomly selected plants revealed 60 %of them had euploid chromosome numbers (2x=4n= 48). Tetraploid plants produced fertile pollen and developed fruits and viable seeds upon self pollination. About two thirds of the tested somatic hybrid plants survived in the winter-cold temperature (Average minimum -1.25°C) to maximum 5.3°C).

Production of somatic hybrids between Daucus carota L. and Nicotiana tabacum

Protoplasts of a kanamycin-resistant (KR, nuclear genome), streptomycin-resistant (SR, chloroplast genome) and chlorophyll-deficient (A1, nuclear genome) Nicotiana tabacum (KR-SA) cell suspension cultures or X-ray-irradiated mesophyll protoplasts of kanamycin- and streptomycin-resistant green plants (KR-SR) were fused with protoplasts of a cytoplasmic male-sterile (CMS) Daucus carota L. cell suspension cultures by electrofusion. Somatic hybrid plants were selected for kanamycin resistance and the ability to produce chlorophyll. Most of the regenerated plants had a normal D. carota morphology. Callus induced from these plants possessed 23-32 chromosomes, a number lower than the combined chromosome number (66) of the parents, and were resistant to kanamycin, but they segregated for streptomycin resistance, which indicated that N. tabacum chloroplasts had been eliminated. Genomic DNA from several regenerated plants was analyzed by Southern hybridization for the presence of the neomycin phosphotransferase gene (NPTII); all of the plants analyzed were found to contain this gene. Mitochondrial (mt) DNA was analyzed by Southern hybridization of restriction endonuclease digests of mtDNA with two DNA probes, PKT5 and coxII. The results showed that the two plants analyzed possessed the mitochondria of D. carota. These results demonstrate that the regenerated plants are interfamilial somatic hybrids.

Production of somatic hybrids between frost tolerantSolanum commersonii andS. Tuberosum: Protoplast fusion, regeneration and isozyme analysis

Mesophyll protoplasts ofSolanum commersonii, a frost tolerant wild species not crossable with the cultivated potato, were fused with either dihaploid or tetraploid S.tuberosum. Protoplasts were aggregated by means of alternating current (AC) or polyethylene glycol (PEG), and electrofused with three direct current (DC) pulses. The treatments with PEG/DC generally resulted in very low heterofusion frequency and protoplast viabiity. On the other hand, AC/DC fusion conditions were optimized by increasing the fusion density of protoplasts and adding CaCl2 to fusion medium. When a density of 4.8 × 105 protoplasts ml−1 was used in the fusion medium containing 0.2 mM Ca++, AC/DC treated protoplasts showed heterofusion frequencies and plating efficiencies of about 10 and 3%, respectively. Fast growing calli from AC/DC fusion experiments were further cultured for regeneration. Fifty-seven plants were regenerated and clonedin vitro as shoot cultures. Compared to parents they showed heterotic vigor and could be identified as hybrids, based on isozyme analysis.

Production of somatic hybrids between frost-tolerant Solanum commersonii and S. tuberosum: characterization of hybrid plants

Somatic fusion of mesophyll protoplasts was used to produce hybrids between the frost-tolerant species Solanum commersonii (2n=2x=24) and dihaploid S. tuberosum (2n=2x=24). This is a sexually incompatible combination due to the difference in EBN (Endosperm Balance Number, Johnston et al. 1980). Species with different EBNs as a rule are sexually incompatible. Fifty-seven hybrids were analysed for variation in chromosome number, morphological traits, fertility and frost tolerance. About 70% of the hybrids were tetraploid, and 30% hexaploid. Chloroplast counts in stomatal guard cells revealed a low frequency of cytochimeras. The frequency of aneuploids was relatively higher at the hexaploid level (hypohexaploids) than at the tetraploid level (hypotetraploids). The somatic hybrids were much more vigorous than the parents, and showed an intermediate phenotype for several morphological traits and moderate to profuse flowering. Hexaploid hybrid clones were less vigorous and had a lower degree of flowering than the tetraploid hybrid clones. All of the hybrids were female fertile but male sterile except for one, which was fully fertile and self-compatible. Many seeds were produced on the latter clone by selfing and on the male-sterile clones by crossing. The somatic hybrid plants showed an introgression of genes for frost tolerance and an adaptability to cold from S. commersonii. Therefore, the use of these somatic hybrids in breeding for and in genetic esearch on frost tolerance and cold-hardening is suggested.

Production of somatic hybrids between Nicotiana benthamiana and N. tabacum and their resistance to aphids

In order to introduce aphid resistance into tobacco, somatic hybrids between tobacco and aphid resistant Nicotiana benthamiana were produced. The hybrids which were proved to be true hybrids on the basis of electrophoretic analysis of ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) and restriction fragment length polymorphism (RFLP) analysis showed significant resistance to aphids. In the leaf surface lipids of the hybrids, lipids specific to N. benthamiana were detected with those specific to tobacco. Five out of 22 of the hybrids were fertile in self-pollination.

Production of Somatic Hybrids between Southern Type Chinese Cabbage 'Kenshin' and Cabbage 'Yoshin' and Their Flowering Characteristics.

Production of Somatic Hybrids between Southern Type Chinese Cabbage 'Kenshin' and Cabbage 'Yoshin' and Their Flowering Characteristics.