Somatic hybrids between Brassica oleracea and B. campestris: selection by the use of iodoacetamide inactivation and regeneration ability

Summary An efficient procedure for obtaining somatic hybrids between B. oleracea and B. campestris has been developed. Hypocotyl protoplasts of B. oleracea were fused with mesophyll protoplasts from three different varieties of B. campestris by the polyethylene glycoldimethylsulfoxide method. The selection of somatic hybrids utilized the inactivation of B. oleracea protoplasts by iodoacetamide (IOA) and the low regeneration ability of B. campestris. The efficiency of recovery of somatic hybrids depended upon the IOA concentration, and when 15 mM IOA was used, 90% of the regenerated plants were found to be hybrid. The somatic hybrids were examined for i) leaf morphology, ii) leucine aminopeptidase (LAP) isozyme and iii) chromosome number. All the hybrids had intermediate leaf morphology and possessed LAP isozymes of both parental species. The chromosome analysis revealed a considerable variation in chromosome number of somatic hybrids, showing the occurrence of multiple fusion and chromosome loss during the culture. Some of the hybrids flowered and set seeds.     

Distribution and changes of reserve materials in cotyledon cells of Panax ginseng related to direct somatic embryogenesis and germination

Cotyledon explants from zygotic embryos of Panax ginseng produced somatic embryos on Murashige and Skoog basal medium without growth regulators. Somatic embryos developed directly from epidermal cells at the cotyledon base. Somatic embryos were always formed from the side of the cotyledon opposite to the one attached to the medium surface regardless of cotyledon orientation. The frequency of somatic embryo formation from the abaxial epidermis (66%) was much higher than that from the adaxial epidermis (12%). Differences in embryogenic response were likely related to cell structure. Abaxial epidermal cells were filled with reserve materials (lipid bodies), while adaxial epidermal cells were devoid of any prominent reserves. During germination, the reserve materials in the cells of the cotyledons disappeared rapidly. At the same time, the competency of somatic embryo formation from cotyledon explants declined rapidly to zero. Upon culture of the cotyledon explants (for somatic embryo induction), lipid bodies slowly disappeared, but starch grains accumulated prominently. Reserve materials disappeared after commencement of embryogenic cell division. During germination, lipid bodies rapidly disappeared, and chloroplasts developed instead of starch grains. Received: 29 January 1997 / Revised version received: 16 April 1997 / Accepted: 9 May 1997     

Production of somatic hybrids by electrofusion in Solanum

Summary Conditions are described for large scale electrofusion of mesophyll protoplasts of dihaploid S. tuberosum with those of diploid S. brevidens. Overall fusion frequencies of 20%–30% were achieved, and following fusion, large numbers of protoplast-derived calli were obtained. Putative somatic hybrid plants were selected from the regenerated shoots by examining their morphological characteristics. Twenty-one somatic hybrids were confirmed by isoenzyme analysis and six somatic hybrids were further confirmed by Southern hybridization. Tetraploid hybrids were obtained, but cytogenetic studies indicated that more of the regenerated hybrids were hexaploid than had previously been found following chemical fusion of the same partners. Some advantages of electrofusion over chemical fusion are discussed.     

Repetitive somatic embryogenesis in Medicago truncatula ssp. Narbonensis and M. truncatula Gaertn cv. Jemalong

Medicago truncatula ssp Narbonensis and four genotypes of M. truncatula Gaertn cv. Jemalong were tested for their somatic embryogenesis potential using a two-step protocol. In the first step, embryogenic callus was induced in folioles isolated from shoots grown in vitro and cultured on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid and zeatin. In the second step, somatic embryos were allowed to develop from the induced callus in MS growth-regulator-free medium. Individual somatic embryos were then isolated and transferred again to growth regulator free medium where they formed secondary somatic embryos in repetitive cycles. Conversion of somatic embryos into plantlets was achieved by isolating late-torpedo-phase somatic embryos with distinct cotyledons and reculturing them onto MS growth regulator free medium. The system of repetitive somatic embryogenesis in M. truncatula described here represents a permanent source of embryogenic material that can be used for the genetic modification of this species. Received: 7 August 1997 / Revision received: 22 December 1997 / Accepted: 20 January 1998     

Production and characterization of somatic hybrid plants between leek (Allium ampeloprasum L.) and onion (Allium cepa L.)

 Results are reported on the production and characterization of somatic hybrids between Allium ampeloprasum and A. cepa. Both symmetric and asymmetric protoplast fusions were carried out using a polyethylene-based mass fusion protocol. Asymmetric fusions were performed using gamma ray-treated donor protoplasts of A. cepa and iodoacetamide-treated A. ampeloprasum protoplasts. However, the use of gamma irradiation to eliminate or inactivate the donor DNA of A. cepa proved to be detrimental to the development of fusion calli, and thus it was not possible to obtain hybrids from asymmetric fusions. The symmetric fusions yielded a high number of hybrid calli and regenerated plants. The analysis of the nuclear DNA composition using interspecific variation of rDNA revealed that most of the regenerated plants were hybrids. Flow cytometric analysis of nuclear DNA showed that these hybrid plants contained a lower DNA content than the sum of the DNA amounts of the parental species, suggesting that they were aneuploid. A shortage of chromosomes in the hybrids was confirmed by genomic in situ hybridization. Chromosome counts in metaphase cells of six hybrids revealed that these plants lacked 2–7 leek chromosomes. One hybrid showed also the loss of onion chromosomes. The hybrids had an intermediate phenotype in leaf morphology. The application of these somatic hybrids in breeding is discussed. Received: 7 April 1997 / Accepted: 10 September 1997     

Chromosomes are eliminated in the symmetric fusion between <Emphasis Type="Italic">Arabidopsis </Emphasis><Emphasis Type="Italic">thaliana</Emphasis> L. and <Emphasis Type="Italic">Bupleurum scorzonerifolium</Emphasis> Willd.

In order to investigate chromosome elimination in symmetric somatic hybridization between Bupleurum scorzonerifolium and Arabidopsis thaliana, protoplasts were isolated from suspension cultures of both A. thaliana and B. scorzonerifolium parents. Biparental protoplasts were mixed at a rate of 1.5:1 and fused with PEG-method. After protoplast fusion, the products were cultured in the P5 liquid medium for microcallus formation. Single cell lines formed from microcalli after subculturing on the MB1 (Xia and Chen, Plant Sci 120:197–203, 1996) solid medium. The putative somatic hybrid cell lines were identified by cytological and molecular analysis. Of the 132 somatic cell lines generated, 16 were identified as somatic hybrids, with the phenotypes resembled B. scorzonerifolium parent. These hybrids showed a complete set of B. scorzonerifolium chromosome and 0–2 small chromosome(s) of A. thaliana. A few of them showed nuclear and cytoplasmic SSR fragments of A. thaliana. These hybrid cell lines could differentiate to green spots, buds/leaves through complementation of regeneration ability. The chromosomes elimination of A. thaliana was discussed. Wang Minqin and Zhao Junsheng contributed equally to the work.     

High-efficiency plant production via direct somatic single embryogenesis from preplasmolysed cotyledons of Panax ginseng and possible dormancy of somatic embryos

Cotyledon explants of immature ginseng zygotic embryos cultured on Murashige and Skoog medium lacking growth regulators formed somatic embryos directly, most in a multiple state, fused together and to the parent cotyledon explants. When the cotyledon explants of ginseng were pretreated with 1.0 m sucrose for 24–72 h, all the somatic embryos developed individually from all surfaces of the cotyledons and the number of somatic embryos per explant was enhanced fourfold. Histological observation revealed that all the single somatic embryos from preplasmolysed cotyledons originated from epidermal single cells, whereas all the multiple embryos from cotyledons without pretreatment originated from epidermal and subepidermal cell masses. When the somatic embryos matured to the cotyledonary stage, further growth ceased and they remained white, probably indicating dormancy. Gibberellic acid (GA₃) (over 1.0 mg/l) or chilling treatment (–2°C for over 8 weeks) were prerequisites for the germination of somatic embryos. Ultrastructural observation revealed that the cotyledon cells of somatic embryos without chilling or GA₃ treatment contained numerous lipid reserves, dense cytoplasm, proplastids and non-activated mitochondria, whereas the cotyledon cells of somatic embryos after chilling or GA₃ treatment were highly vacuolated and contained well-developed chloroplasts and active-state mitochondria enclosing numerous cristae, indicating that in-vitro-developed somatic embryos of P. ginseng may be dormant after maturing in a manner similar to zygotic embryos. Received: 8 July 1998 / Revision received: 31 August 1998 / Accepted: 23 September 1998     

Induction of somatic embryogenesis and plant regeneration from cotyledon and hypocotyl explants of <Emphasis Type="Italic">eruca sativa</Emphasis> mill

Summary A protocol was developed for high frequency somatic embryogenesis and plant regeneration from cotyledon and hypocotyl explants of Eruca sativa. Explants grown on Murashige and Skoog (MS) medium supplemented with 4.52 μM 2,4-D formed embryogenic callus after 4 wk of culture. Secondary somatic embryos were also produced from primary somatic embryos on MS medium containing 0.56 μM 2,4-D. Somatic embryos developed into mature embryos on MS medium in the presence of 45 gl⁻¹ polyethylene glycol. After desiccation, somatic embryos developed into plantlets by culturing the mature somatic embryos on 1/2 x MS medium containing 0.24 μM indole-3-butyric acid.     

Hybridisation experiments with protoplasts from chlorophyll-deficient mutants of some Solanaceous species

Following fusion between protoplasts from two different chlorophyll-deficient diploid mutants of Datura innoxia Mill. it was possible to select 33 green hybrid calli on agar culture medium. Half of the somatic hybrids gave rise to leaves and some to shoots. The chromosome number of 20 somatic hybrids was determined: five were tetraploid, eight hexaploid, three octoploid, and four showed an aneuploid chromosome number. After transfer of the shoots of the five tetraploid hybrids to soil they developed roots. In control experiments in which protoplasts of the two mutants were cultured either as a mixture without being treated with the fusion agent, or cultured separately, no green callus could be obtained. Similar experiments involving protoplasts from one chlorophyll-deficient mutant of Datura innoxia, on the one hand, and those from similar mutants of Nicotiana sylvestris Spegazz. et Comes and Petunia hybrida, on the other, yielded no green somatic hybrid although hybrid protoplasts could be detected.     

Regeneration of plants from protoplasts of Capsella bursa-pastoris and somatic hybridization with rapid cycling Brassica oleracea

Fertile rooted plantlets were recovered from leaf mesophyll protoplasts of Capsella bursa-pastoris. Protoplasts cultured over a feeder layer of Brassica napus cells produced 221 colonies, 7 of which regenerated multiple plantlets. The nuclear DNA content of most regenerates varied from 0.89 to 1.0 pg/nucleus, close to the value for seed-grown C. bursa-pastoris (0.94±0.03 pg/nucleus). Two regenerants had a tetraploid DNA content (1.8– 2.0 pg). Plants with a DNA content close to Capsella produced seeds, both in vitro and in soil. Intertribal somatic hybrids were obtained by polyethylene glycol-mediated fusion of untreated C. bursa-pastoris protoplasts with iodoacetate-treated protoplasts of rapid-cycling B. oleracea. Plants were confirmed as somatic hybrids by isozyme and RAPD analysis. The nuclear DNA content of the hybrids ranged from 3.2 to 6.4 pg, higher than the sum of the parental genomes. One of two hybrids tested was resistant to Alternaria brassicicola, like the Capsella fusion partner. Hybrids rooted easily and produced sterile flowers when transplanted to soil. Received: 13 April 1998 / Revision received: 25 August 1998 / Accepted: 31 August 1998     

Molecular analysis of the nuclear organellar genotype of somatic hybrid plants between tomato (Lycopersicon esculentum) and Lycopersicon chilense

Summary Somatic hybrid plants were recovered following fusion of leaf mesophyll protoplasts isolated from tomato (Lycopersicon esculentum) cultivar UC82 with protoplasts isolated from suspension cultured cells of L. chilense, LA 1959. Iodoacetate was used to select against the growth of unfused tomato protoplasts. Two somatic hybrids were recovered in a population of 16 regenerants. No tomato regenerants were recovered; all of the non-hybrid regenerants were L. chilense. The L. chilense protoplast regenerants were tetraploid. The hybrid nature of the plants was verified using species-specific restriction fragment length polymorphisms for the nuclear, chloroplast and mitochondrial genomes. The somatic hybrids had inherited the chloroplast DNA of the tomato parent, and portions of the mitochondrial DNA of the L. chilense parent. The somatic hybrids formed flowers and developed seedless fruit.     

Transfer of amiprophosmethyl resistance from a Nicotiana plumbaginifolia mutant by somatic hybridisation

Transfer of resistance to the phosphorothioamidate herbicide, amiprophosmethyl (APM), from the β-tubulin mutant of Nicotiana plumbaginifolia to the interspecific N. plumbaginifolia (+) N. sylvestris and to the intertribal N. plumbaginifolia (+) Atropa belladonna somatic hybrids has been demonstrated. Transfer to the recipient species was accomplished by: (1) symmetric hybridisationand (2) asymmetric hybridisation using γ-irradiation of donor protoplasts. Cytogenetic analysis confirmed the hybrid origin of the hybrids obtained. It was established that most of them typically inherited no more than three donor chromosomes, although it was possible to obtain symmetric hybrids in the case of symmetric fusion. Immunofluorescent microscopy analysis has shown that protoplasts of the mutant, and of the N. plumbaginifolia (+) N. sylvestris and N. plumbaginifolia (+) A. belladonna hybrids, retained the normal structure of interphase microtubule (MT) arrays and mitotic figures after treatment with 5 μM APM, whereas MTs of protoplasts of the recipients were destroyed under these conditions. It was also shown that hybrid clones contained an altered β-tubulin isoform originating from the N. plumbaginifolia mutant. The selected hybrid clones were characterised by cross-resistance to trifluralin, a dinitroaniline herbicide with the same mode of anti-MT action. Some of the somatic hybrids which could flower were fertile. It was established that seeds of some fertile hybrids were able to germinate in the presence of 5 μM APM. The results obtained thus support the conclusion that the technique of somatic hybridisation, especially asymmetric fusion, can be used to transfer APM resistance from the N. plumbaginifolia mutant to different (related and remote) plant species of the Solanaceae, including important crops. Received: 22 December 1997 / Accepted: 27 July 1999     

Comparison of UV light, X-ray and restriction enzyme treatment as tools in production of asymmetric somatic hybrids between Brassica napus and Arabidopsis thaliana

 Asymmetric somatic hybrids between Brassica napus (receptor) and Arabidopsis thaliana (donor) have been produced by three different methods supposed to induce asymmetry. The donor protoplasts were either UV- or X-irradiated, or the mixture of protoplasts was treated with the restriction enzyme PvuII immediately before fusion. The genome composition of the hybrids was analysed with Southern blot hybridisations using 15 different mapped A. thaliana RFLP markers as probes. Both UV- and X-irradiation were found to be efficient treatments for induction of asymmetry in somatic hybrids in a dose-dependent manner. The addition of a restriction enzyme to the protoplast mixture did not have any effect on the frequency of asymmetric hybrids or on the degree of asymmetry in the hybrids produced. UV- and X-irradiation resulted in higher fertility in the hybrids, while PvuII treatment did not have any effect on seed set. A significant positive correlation between degree of asymmetry in different plants and seed set after selfing was detected. Received: 18 December 1997 / Accepted: 7 January 1998     

Metabolic complementation for a single gene function associated with partial and total loss of donor DNA in interspecific somatic hybrids

Summary We report here on the obtainment of interspecific somatic, asymmetric, and highly asymmetric nuclear hybrids via protoplast fusion. Asymmetric nuclear hybrids were obtained after fusion of mesophyll protoplasts from a nitrate reductase-deficient cofactor mutant of N. plumbaginifolia with irradiated (100 krad) kanamycin resistant leaf protoplasts of a haploid N. tabacum. Selection for nitrate reductase (NR) and/or kanamycin (Km) resistance resulted in the production of three groups of plants (NR⁺, NR⁺, Km^R, and NR^-Km^R). Cytological analysis of some hybrid regenerants showed the presence of numerous tobacco chromosomes and chromosome fragments, besides a polyploid N. plumbaginifolia genome (tetra or hexaploid). All the regenerants tested were male sterile but some of them could be backcrossed to the recipient partner. In a second experiment, somatic and highly asymmetric nuclear hybrids were obtained after fusion of mesophyll protoplasts from the universal hybridizer of N. plumbaginifolia with suspension protoplasts of a tumor line of N. tabacum. Selection resulted in two types of colonies: nonregenerating hybrid calli turned out to be true somatic hybrids, while cytological analysis of regenerants obtained on morphogenic calli did not show any presence of donor-specific chromosomes. Forty percent of the hybrid regenerants were completely fertile, while the others could only be backcrossed to the recipient N. plumbaginifolia. Since the gene we selected for is not yet cloned, we were not able to demonstrate the transfer of genetic material at the molecular level. However, since no reversion frequency for the nitrate reductase mutant is known, and due to a detailed cytological knowledge of both fusion partners, we feel confident in speculating that intergenomic recombination between N. plumbaginifolia and N. tabacum has occurred.     

Somatic hybridization in mint: identification and characterization of Mentha piperita (+) M. spicata hybrid plants

 Twenty eight somatic hybrid plants were identified following protoplast fusions between peppermint (Mentha piperita L. cv Black Mitcham), producing high-quality oil, and spearmint (Mentha spicata L. cv Native Spearmint), likewise producing high-quality oil and also possessing resistance to verticillium wilt. Prior to fusion, peppermint protoplasts were subjected to iodoacetic acid to inhibit cell division. Protoplasts of peppermint and spearmint were fused using polyethylene glycol plus DMSO. Fusion products were cultured according to an efficient protoplast-to-plant-cycle protocol developed for peppermint. Using this protocol, iodoacetic acid-treated peppermint protoplasts were not able to divide, whereas untreated spearmint protoplasts had the ability to produce callus but not shoots. Therefore, selection of somatic hybrid calli was based on the presumed capability of hybrid cells to form calli and shoots. Shoots in vitro were initially identified as hybrids using RAPD profiles. Subsequently, observations on morphology, chromosome counts, and Southern-hybridization patterns confirmed their hybrid status. The results of verticillium tests revealed that 18 somatic hybrids were more susceptible than Native Spearmint, while hybrid II-14 had a level of susceptibility intermediate between that of the fusion parents. Oil-analysis of hybrid plants indicated that they all have a GC-profile typical of spearmint oil. Received: 8 February 1997 / Accepted: 9 April 1997     

Optimization of direct somatic embryogenesis from mature zygotic embryos ofPanax ginseng C. A. Meyer

Culture conditions were optimized for somatic embryogenesis ofPanax ginseng. The highest frequency of embryo formation was obtained when tissues were excised from the middle region of the cotyledon segments of zygotic embryos. Only treatment with light could stimulate the formation of single-type somatic embryos, whereas multiple-type somatic embryos and calli were observed under dark conditions. The highest production of somatic embryos was found with an NH₄ ⁺:NO₃ ratio of 21:39. Among the tested media (MS, B5, and SH), maximum formation of somatic embryos was obtained when cotyledon expiants were cultured on an 1% agar MS medium supplemented with 5% sucrose. Regenerated ginseng plantlets were transferred to an autoclaved soil mixture in the greenhouse. These transformants showed no detectable variations in their morphology or growth characteristics compared with the donor plant.     

Somatic hybridization between selected Lycopersicon and Solanum species

Summary Mesophyll protoplasts of an interspecific Lycopersicon esculentum Mill, (tomato) x Lycopersicon pennellii hybrid plant (EP) were fused with callus-derived protoplasts of Solanum lycopersicoides Dun. using a modified PEG/DMSO procedure. The EP plant was previously transformed by Agrobacterium tumefaciens which carried the NPTII and nopaline synthase genes. Protoplasts were plated at 10⁵/ml in modified KM medium and 16 days post-fusion 25 ug/ml kanamycin was added to the culture medium. During shoot regeneration, 212 morphologically similar putative somatic hybrids were delineated visually from kanamycin resistant EP's. Forty-eight shoots, randomly selected among the 212, were further verified as somatic hybrids by their leaf phosphoglucoisomerase heterodimer isozyme pattern. However, the resulting plants were virtually pollen sterile. In a second fusion, mesophyll protoplasts of Solanum melongena (eggplant) were fused with EP callus-derived protoplasts. Using the same fusion and culture procedure, only two dark green calli were visually selected among the pale green parental EP and verified as somatic cell hybrids by several isozyme patterns. These two calli have produced only leaf primordia in one and half years on regeneration medium.Abbreviations ABA abscisic acid - BAP 6 benzylaminopurine - 2,4-D 2,4 dichlorophenoxy acetic acid - DMSO dimethyl sulfoxide - GA₃ gibberellic acid - GOT glutamate oxaloacetate - IAA indoleacetic acid - IBA indolebutyric acid - IDH isocitrate dehydrogenase - MDH malate dehydrogenase - MES morpholinoethane-sulfonic acid - PEG polyethylene glycol - 6-PGDH 6 phosphogluconate dehydrogenase - PGI phosphoglucoisomerase     

Somatic hybrids obtained by asymmetric protoplast fusion between <Emphasis Type="Italic">Musa</Emphasis> Silk cv. Guoshanxiang (AAB) and <Emphasis Type="Italic">Musa acuminata</Emphasis> cv. Mas (AA)

To attempt to introduce genetic information of disease resistance from Musa acuminata cv. Mas (AA) to Musa silk cv. Guoshanxiang (AAB) and obtain somatic hybrids, we developed an asymmetric protoplast fusion with 20% (w/v) polyethylene glycol (PEG). The protoplasts derived from embryogenic suspension cultural cells of cv. Guoshanxiang (AAB) and cv. Mas (AA) were, respectively treated with 1.5 mM iodoacetamide (IOA) and with ultraviolet light (UV) at an intensity of 50 W/m² for 120 s. A total of 47 regenerated green plants were obtained and eight of which were survived in greenhouse. Six of the survived plants were identified as hybrids by RAPD analysis and only three hybrids were retained vigorously in field. The hybrid nature of the three plants was further confirmed according to their ISSR (inter-simple sequence repeat) patterns and the results indicated that they were true somatic hybrids. Chromosome analysis revealed that the three hybrids possessed an aneuploid chromosome number (2n = 34).     

Somatic hybrids of Datura innoxia Mill.+Datura discolor Bernh. and of Datura innoxia Mill.+Datura stramonium L. var tatula L.

Summary Following fusion of protoplasts from a chlorophyll-deficient diploid mutant of Datura innoxia Mill. which can be regenerated to shoots, with green wild-type protoplasts of Datura stramonium L. var. tatula L. which can not, it was possible to isolate 49 green hybrid calli on agar medium. Most of these somatic hybrid calli gave rise to leaves and shoots. The chromosome numbers of the somatic hybrids were determined: 15 were tetraploid (amphidiploid), 24 hexaploid, and the other showed an aneuploid chromosome number.In a similar experiment protoplasts of the Datura innoxia mutant were fused with green wild-type protoplasts of Datura discolor Bernh. which are also not able to be regenerated, four green calli were obtained from which leaves and shoots developed after some transfers on agar medium. Three of them showed the amphidiploid (48) chromosome number, whereas one possessed an aneuploid number of 46 chromosomes.After transfer of rooted shoots to soil flowering plants could be obtained in both combinations. The habits of the somatic hybrids in both combinations were intermediate between the habits of the respective parental plants.Dedicated to my father, Prof. Dr. Theodor Schieder, on the occasion of his 70th birthday.     

Cyclic somatic embryogenesis and efficient plant regeneration from callus of safflower

Efficient plant regeneration through somatic embryogenesis was established for safflower (Carthamus tinctorius L.) cv. NARI-6. Embryogenic calli were induced from 10 to 17-d-old cotyledon and leaf explants from in vitro seedlings. High frequency (94.3 %) embryogenic callus was obtained from cotyledon explants cultured on Murashige and Skoog’s germination (MSG) basal medium supplemented with thidiazuron, 2-isopentenyladenine and indole-3-butyric acid. Primary, secondary and cyclic somatic embryos were formed from embryogenic calli in a different media free of plant growth regulators, however, 100 % cyclic somatic embryogenesis was obtained from cotyledon derived embryogenic calli cultured on MSG. Somatic embryos matured and germinated in quarter-strength MSG medium supplemented with gibberellic acid. Cotyledons with root poles or non root poles were converted to normal plantlets and produced adventitious roots in rooting medium. Rooted plants were acclimatized and successfully transferred to the field.