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

Summary Three sexual generations of the somatic hybrids from Datura innoxia+Datura stramonium (Datura straubii) and from Datura innoxia+Datura discolor (Datura hybrida) were investigated (Schieder, 1978). Self fertility of the somatic hybrids proved to be comparable to that of the parental plant species. The good fertility is attributed to the low number of quadrivalents formed in meiosis I. The growth rate of the somatic hybrids is superior to the parental plant species and also to the autotetraploid somatic hybrids of Datura innoxia. Datura innoxia is the species showing the best growth rate of all species used for the hybridization experiments. In addition, some irregularities in the sexual generations are described.Dedicated to my teacher, Prof. Dr. J. Straub, on the occasion of his 70^th birthday.     

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.     

Isolation of mutants with altered pigments after irradiating haploid protoplasts from Datura innoxia Mill. with X-rays

Summary Ten different mutants with altered pigment patterns were isolated following X-irradiation of approximately 10⁵ haploid protoplasts of Datura innoxia Mill. Seven of the selected strains gave rise to shoots and 3 to leaves only. The mutants were selected from light green or white calli, which had developed 4 weeks after transfer of developing cell clusters onto B₅ agar medium containing 0.5 mg/l BAP (Gamborg et al., 1968). Of the 10 mutant strains 5 were light green, two were yellow, one was pale yellow and one was white. One additional strain does not possess anthocyanin in its stems; a feature chracteristic of the wildtype is the possession of anthocyanin. This strain is able to grow in soil and has now flowered. None of the mutants obtained is haploid. Nine are diploid and the other is tetraploid. The chlorophyll deficient strains can be propagated on B₅ agar medium supplemented with higher concentration of sucrose than normally required for the growth of the wild-type.     

Selection of somatic hybrids after fusion of protoplasts from Datura innoxia Mill. and Atropa belladonna L.

After fusion of protoplasts from a diploid (2n=24) and a tetraploid (4n=48) chlorophyll-deficient mutant of Datura innoxia Mill. with diploid (2n=72) green wild-type protoplasts of Atropa belladonna L. thirteen somatic hybrids could be selected, most of which had already started to produce leaves and shoots. Hybrid calli were recognizable by the production of hairs, typical for Datura innoxia, and the green colour, derived from Atropa belladonna. Further proof for the hybrid nature was furnished by cytological investigations. The metaphase chromosomes of both species are easily distinguishable in their size: chromosomes of Datura innoxia are about twice as large as those of Atropa belladonna. The chromosome numbers of the hybrids varied from ca. 84 to ca. 175.Abbreviations BAP 6-benzylaminopurine - NAA -naphthalene acetic acid     

Alkaloid production by plants regenerated from cultured cells of Datura innoxia

Cellular aggregates in Datura innoxia suspension cultures give rise to large numbers of shoots when such aggregates are cultured in the light on an auxin-free agar medium supplemented with kinetin. These shoots form roots on a kinetin-free medium to develop into complete plants. Most of the regenerated plants are diploid, and the frequencies of ancuploid or polyploid plants are much lower than might be expected from the distribution of chromosome number in the cultured cells. During root ditterentiation and plant development, scopolamine synthesis is initiated and there is a progressive increase in the alkaloid content. Consequently, the general pattern of alkaloid composition is restored to a normal state in the majority of the regenerated plants including aneuploid or polyploids. Nevertheless, some of the plants show an abnormal expression in alkaloid metabolism, such as the complete hydrolysis of scopolamine in the dried leaves.     

Comparison of somatic and sexual incompatibility between Datura innoxia and Atropa belladonna

After protoplast fusion somatic hybrid calli were obtained by complementation selection between an albino mutant of Datura innoxia and the wildtype of Atropa belladonna (Krumbiegel and Schieder, 1979. Planta 145, 371–375). In the present study experiments are described concerning leaf and shoot induction on several media supplemented with different combinations and concentrations of hormones. Except for fleshy leaves and embryos, no well-formed shoot could be obtained. However, under standard culture conditions after one and a half years, one line produced numerous green shoots, showing a reduced number of chromosomes from Atropa belladonna. The loss of some chromosomes decreased the degree of somatic incompatibility. The additional appearance of shoots with albino sectors, of total albino shoots, and of green shoots showing a different phenotype, demonstrated that the elimination of the chromosomes occurred not only once, but several times. At least one shoot nearly stable in chromosome content and green subline could be obtained possessing only 6 chromosomes of Atropa belladonna and the original chromosome number of Datura innoxia. Experiments were carried out to test the feasibility of producing sexual hybrids through in vivo and in vitro methods by cross pollination. However, no embryos, seeds, or plantlets were obtained, thus demonstrating that protoplast fusion is the only possibility for obtaining hybrids between these two species.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlor-phenoxyacetic acid - IAA indoleacetic acid - NAA -naphtaleneacetic acid - SDS sodiumdodecylsulfate     

Asymmetric somatic hybridization between wheat (Triticum aestivum) and Avena sativa L.

Protoplasts from cell suspensions of young-embryo-derived calli, which were nonregenerable for long-term subculture and protoplasts from embryogenic calli with the regeneration capacity of 75% of the same wheat Jinan 177, were mixed as recipient. Protoplasts from embryogenic calli of Avena sativa (with the regeneration capacity of less than 10%) irradiated with UV at an intensity of 300 μW/cm² for 30 s, 1 min, 2 min, 3 min, 5 min were used as the donor. Protoplasts of the recipient and the donor were fused by PEG method. Many calli and normal green plants were regenerated at high frequency, and were verified as somatic hybrids by chromosome counting, isozyme, 5S rDNA spacer sequence analysis and GISH (genomic in situ hybridization). Fusion combination between protoplasts either from the cell suspensions or from the calli and UV-treated Avena sativa protoplasts could not regenerate green plants.     

Genetic evidence for the hybrid nature of somatic hybrids from Datura innoxia Mill.

The hybrid nature of tetraploid somatic hybrids of two genetically different chlorophyll-deficient mutants from Datura innoxia Mill. was demonstrated with the aid of anther culture. Green and chlorophyll-deficient androgenetic lines could be regenerated from the pollen grains.     

Aluminum elicits tropane alkaloid production and antioxidant system activity in micropropagated Datura innoxia plantlets

One of the methods which enhances production of plant secondary metabolites including alkaloids is the use of abiotic elicitors in vitro. The aim of the study was the employment of aluminum (Al) as an abiotic elicitor in such concentrations, which were not stressful for the plant, to avoid intense growth limiting, but could elicit tropane alkaloid biosynthesis. The in vitro propagated plantlets of Datura innoxia were exposed to different concentrations of AlCl₃ (0, 25, 75 and 225 μM) to determine its effects on hyoscyamine and scopolamine contents in roots and shoots, which were analyzed by HPLC. Antioxidant enzyme activities were determined by spectrophotometer. Results showed that aluminum reduced shoots’ and roots’ fresh weights and the reduction in roots were more than the shoots. In addition, Al had significant positive effects on hyoscyamine and scopolamine contents especially in roots of the plantlets and AlCl₃ caused ROS production in shoots. These findings suggest that aluminum can likely elicit the tropane alkaloid biosynthesis in D. innoxia plantlets in vitro.     

Regeneration of somatic hybrid plants formed between Lycopersicon esculentum and L. pennellii

Summary Somatic hybrid plants have been regenerated following polyethylene glycol mediated fusion of leaf mesophyll protoplasts from tomato and protoplasts from Lycopersicon pennellii callus. Three different cultivars of tomato were used as sources of protoplasts: Early Girl, Manapal, and UC82B. Fusions were performed between protoplasts of these tomato cultivars and protoplasts of L. pennellii, and between protoplasts of the cultivars and protoplasts of L. pennellii that had been exposed to 3 or 6 krads of gamma radiation. Somatic hybrid plants were identified on the basis of heterozygous isozyme banding patterns, and leaf and flower morphology. Somatic hybrid plants were regenerated following fusion of tomato protoplasts with either untreated or irradiated L. pennellii protoplasts. All were heterozygous for isozyme loci on five different chromosomes. Regenerated somatic hybrids showed inheritance of either or both parental chloroplast genomes, but predominantly the L. pennellii mitochondrial genome. The regenerated somatic hybrid plants exhibited reduced fertility, less than 20% viable pollen. A total of 34 somatic hybrid calli were identified. Of these, 21 regenerated shoots, and 7 produced seed following manual pollinations.     

Intergeneric somatic hybrid plantlets between Dianthus barbatus and Gypsophila paniculata obtained by electrofusion

Hypocotyl-derived protoplasts of Dianthus barbatus that had been pretreated with iodoacetamide were fused electrically with cell suspension culture-derived protoplasts of Gypsophila paniculata that could divide to form callus but could not regenerate shoots under the culture conditions used in this study. Electrofusion-derived calli which produced shoots were selected as putative somatic hybrids, and plantlets were subsequently regenerated from 2 of these selected calli. These plantlets, which in vitro produced flowers precociously, were identified as intergeneric somatic hybrids by nuclear ribosomal DNA analysis. Normal plants have not been established up to the present.     

Somatic hybrid plants between the forage legumes Medicago sativa L. and Medicago arborea L.

Interspecific somatic hybrid plants were obtained by symmetrical electrofusion of mesophyll protoplasts of Medicago sativa with callus protoplasts of Medicago arborea. Somatic hybrid calli were picked manually from semi-solid culture medium after they were identified by their dual color in fluorescent light. Twelve putative hybrid calli were selected and one of them regenerated plants. The morphogenesis of the somatic hybrid calli was induced by the synthetic growth regulator 1,2 benzisoxazole-3-acetic acid. Somatic hybrid plants showed intensive genome rearrangements, as evidenced by isozyme and RFLP analysis. The morphology of somatic hybrid plants was in general intermediate between the parents. The production of hybrids by protoplast fusion between sexually incompatible Medicago species is related to the in vitro respon siveness of the parental protoplasts. The possibility of using somatic hybrid plants in alfalfa breeding is discussed.     

Plantlet regeneration from somatic hybrids of rice (Oryza sativa L.) and barnyard grass (Echinochloa oryzicola Vasing)

Summary Somatic hybridization of rice (Oryza sativa L.) and barnyard grass (Echinochloa oryzicola), a close relative of barnyard millet, was attempted using electrofusion and a new culture method developed for rice protoplasts (Kyozuka et al. 1987) to incorporate some of the agronomically important characters of the latter species into rice. Selection of hybrids was based on inactivation of rice protoplasts by iodoacetamide and the inability of barnyard grass protoplasts to divide. A total of 166 calli were identified as hybrids by isozyme and chromosome analyses. Hybrid calli were highly morphogenic, and 44 shoots were obtained. Most of them, however, were abnormal, and nine grew to plantlets whose morphology was distinct from that of either parent. Our study clearly demonstrates the totipotency of protoplasts in graminaceous monocots.     

Protoplast fusion between Lycopersicon esculentum and L. peruvianum-complex: somatic embryogenesis, plant regeneration and morphology

Somatic hybrids were obtained by polyethylene glycol fusion of cotyledon protoplasts of Lycopersicon esculentum Mill. cv. Kyoryokutoko treated with iodoacetamide (IOA) and suspension-culture-derived protoplasts of L. peruvianum (PI270435) or L. chilense (PI128652). The hybrids were selected by a multiple-step selection procedure relying on the different colors of the fusion partners, IOA treatment of cotyledon protoplasts, and the use of a culture medium which only allowed cotyledon protoplasts to regenerate. The somatic embryos were derived from greenish calli that formed from the fusion mixtures, developed progressively through the globular, heart, and torpedo stages, and finally formed complete plantlets. The excised torpedo-stage embryos could be propagated on a modified medium. The morphology of the somatic hybrids were intermediate to their donor partners, and chromosome observations indicated that the hybrids were tetraploid, hexaploid, and aneuploid. Received: 24 July 1997 / Revision received: 4 November 1997 / Accepted: 2 December 1997     

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     

Asymmetric somatic hybridization between Triticum aestivum L. and Haynaldia villosa Schur

Suspension cell-derived protoplasts of wheat, inactivated with different concentrations (0-2.5mol/L) of IOA, were fused by PEG method with the Haynaldia villosa protoplasts which originated from the calli 4-5d after subculture and were irradiated with 60Co-γ ray. Cell colonies, calli or regenerated plants were obtained from different combinations of fusion. The calli and plants were verified to be hybrids by chromosome counting, isozyme analysis and morphological inspection.     

Asymmetric somatic hybrid plants between an interspecific Lycopersicon hybrid and Solanum melongena

Summary Asymmetric somatic hybrid plants were obtained by a modified PEG/DMSO fusion procedure between protoplasts derived from suspension cells of an interspecific tomato hybrid, Lycopersicon esculentum x L. pennellii, and mesophyll protoplasts of Solanum melongena, eggplant. The tomato hybrid was previously transformed with Agrobacterium tumefaciens and contained the kanamycin-resistance marker gene. Prior to fusion, the donor protoplasts of the tomato hybrid were gamma irradiated at 9.0 krad. Thus, non-division of irradiated tomato hybrid protoplasts coupled with kanamycin sensitivity of eggplant enabled selection of somatic cell hybrids. Forty-nine calli selected post-fusion regenerated leaf-like structures in the presence of 50 mg/l kanamycin. However, only four of the 49 calli regenerated intact shoots which rooted in the presence of 50 mg/l kanamycin and were later transferred to the greenhouse. Analysis of phosphoglucoisomerase and peroxidase isozymes, and Southern hybridization with a nuclear-specific pea 45 S ribosomal RNA gene confirmed somatic hybrid status. Cytology revealed that the four hybrid plants had chromosome numbers of 45, 60, 42 and 57, respectively; they were all sterile.     

Fertile asymmetric somatic hybrids between Lycopersicon esculentum Mill. and Lycopersicon peruvianum var. dentatum Dun.

Summary Thirteen nuclear asymmetric hybrids were regenerated under selective conditions following fusion of chlorophyll-deficient protoplasts from cultivated tomato (Lycopersicon esculentum Mill.) and -(-irradiated protoplasts from the wild species Lycopersicon peruvianum var. dentatum Dun. All hybrid plants were classified as being asymmetric based on morphological traits, chromosome numbers and isozyme patterns. The majority of the hybrids inherited Lycopersicon peruvianum var. dentatum chloroplasts. Mitochondrial DNA analysis revealed mixed mitochondria populations deriving from both parents in some of the hybrids and rearranged mitochondrial DNA in others. The asymmetric hybrids express some morphological traits that are not found in either of the parental species. Fertile F₁ plants were obtained after self-pollination of the asymmetric hybrids in four cases. The results obtained confirm the potential of asymmetric hybridization as a new source of genetic variation, and as a method for transferring of a part of genetic material from donor to recipient, and demonstrate that it is possible to produce fertile somatic hybrids by this technique.     

Production and identification of somatic hybrids between Solanum tuberosum and S. papita by using the rolC gene as a morphological selectable marker

A successful hybridization of a diploid clone of Solanum tuberosum with a rolC-transgenic, diploid S. papita clone is reported. By using leaf expiants of this S. papita clone, which after transformation expressed kanamycin resistance, intact protoplasts were obtained, but these protoplasts did not develop to microcalli or regenerate to mature plants. However, protoplasts of the S. tuberosum clone showed a high capacity to regenerate plants from isolated protoplasts. On a medium containing Kanamycin only calli regenerated to plants, which revealed a rolC phenotype (reduced apical dominance with a large number of adventitious shoots and a pale green color of leaves) and later on turned out to be true hybrids. Self fusions of S. papita never developed to microcalli and those of S. tuberosum ceased to develop on the kanamycin-containing medium. Identification of somatic hybrids was done by RFLP and RAPD analysis. In the greenhouse, out of four selected hybrids only FK3.1 was successfully crossed with two standard S. tuberosum varieties (Datura, Desirée). Out of all the seeds germinated, only rolC-negative F¹ seedlings were further characterized. Within the seedling population obvious differences were evident in respect of the S. papita and S. tuberosum characteristics.     

Fertile fruit trees obtained by somatic hybridization: navel orange (Citrus sinensis) and Troyer citrange (C. sinensis x Poncirus trifoliata)

Summary Nucellar cell suspension protoplasts of navel orange (Citrus sinsensis Osb.) were chemically fused with mesophyll protoplasts of Troyer citrange (C. sinensis x Poncirus trifoliata) and cultured in hormone-free Murashige and Tucker medium containing 0.6 M sucrose. Two types of plant were regenerated through embryogenesis. One type showed intermediate mono-and difoliate leaves and the other types was identical to Troyer citrange. The regenerated plants with intermediate morphology were demonstrated by chromosome counts and rDNA analysis to be amphidiploid somatic hybrids. Five clones of these somatic hybrids were grafted in the field. After 4 years, they set flowers having a morphology intermediate between those of the two parents. The pollen grains showed high stainability and sufficient germinability, and were larger than those of Troyer citrange. The fruits of the somatic hybrids were large and spherical with thick rinds. Most of them contained seeds with normal germinability. These results indicate that somatic hybridization is a useful tool for Citrus breeding.