Transmission of paternal chloroplasts in Nicotiana

Summary Transmission of paternal chloroplasts was observed in Nicotiana, considered to inherit organelles in a strictly maternal way. Plants carrying streptomycin resistant plastids were used as pollen donors. Cell lines with paternal plastids in the offspring were selected as green (resistant) sectors on calli induced from the seedlings on streptomycin-containing media. The presence of paternal plastids in the regenerated plants was confirmed by restriction analysis. In the Nicotiana plumbaginifolia xN. plumbaginifolia Np(SR1)3 and the N. plumbaginifolia Np(gos)29 xN. tabacum SR1 crosses 2.5% and 0.07% of the offspring were found to contain paternal (tabacum) plastids, respectively. These plants, however, carried maternal mitochondria exclusively. This sexual cybridization method offers a simple way to transfer chloroplasts solely, a goal not accessible by protoplast fusion.     

Effect of radiation dosage on efficiency of chloroplast transfer by protoplast fusion in Nicotiana

Chloroplasts of Nicotiana tabacum SR1 were transferred into Nicotiana plumbaginifolia by protoplast fusion. The protoplasts of the organelle donor were irradiated with different lethal doses using a ⁶⁰Co source, to facilitate the elimination of their nuclei from the fusion products. After fusion induction, clones derived from fusion products and containing streptomycin-resistant N. tabacum SR1 chloroplasts were selected by their ability to green on a selective medium. When N. tabacum protoplasts were inactivated by iodoacetate instead of irradiation, the proportion of N. plumbaginifolia nuclear segregant clones was low (1–2%). Irradiation markedly increased this value: Using 50, 120, 210 and 300 J kg^-1 doses, the frequency of segregant clones was 44, 57, 84 and 70 percent, respectively. Regeneration of resistant N. plumbaginifolia plants with SR1 chloroplasts indicated that plastids can be rescued from the irradiated cells by fusion with untreated protoplasts. Resistant N. plumbaginifolia plants that were regenerated (43 clones studied) had diploid (2n = 2X = 20) or tetraploid chromosome numbers and were identical morphologically to parental plants. The absence of aneuploids suggests that in these clones irradiation resulted in complete elimination of the irradiated N. tabacum nuclei. Resistance is inherited maternally (five clones tested). The demonstration of chloroplast transfer and the presence of N. tabacum plastids in the N. plumbaginifolia plants was confirmed by chloroplast DNA fragmentation patterns after EcoRI digestion.     

Transmission of paternal chloroplasts in tobacco (Nicotiana tabacum)

Medgyesy et al. (1986, Mol. Gen. Genet. 204, 195–198) have described in Nicotiana plumbaginifolia and in an interspecific cross involving N. plumbaginifolia and N. tabacum a procedure for selecting cell lines derived from seedlings carrying paternal chloroplasts by taking advantage of a plastid-encoded mutation which confers resistance to streptomycin. We have extended their demonstration of occasional transmission of chloroplasts through pollen to the case of an intraspecific cross in N. tabacum. The line used as maternal parent, ITB19(sua), displayed a cytoplasmic male sterility due to the presence of a cytoplasm originating from N. suaveolens. The line used as paternal parent, SR1, was fertile and possessed mutant chloroplasts conferring resistance to streptomycin. From cell lines derived from 204 seedlings, three were regenerated into streptomycin-resistant buds. The plants derived from these three clones were male-sterile. Their progeny, after crossing with a wild type tobacco line, XHFD8, was resistant to streptomycin. Tests of resistance of the seedlings to tentoxin and restriction analyses of the chloroplast DNA indicated that two clones still had the maternal chloroplasts and were thus probably new streptomycin-resistant mutants, whereas the third one had acquired the chloroplasts of the paternal parent, but had retained the mitochondria of the maternal parent.Abbreviations cp-DNA chloroplast DNA - mt-DNA mitochondrial DNA - Np Nicotiana plumbaginifolia - Nt Nicotiana tabacum     

Asymmetric hybridization in Nicotiana by fusion of irradiated protoplasts

Summary Mesophyll protoplasts of a kanamycin-resistant, nopaline-positive Nicotiana plumbaginifolia seed line were inactivated by -irradiation and electrically fused with unirradiated mesophyll protoplasts of N. tabacum. Hybrids were selected on kanamycin and regenerated. Genetic material from N. plumbaginifolia was detected in these plants by the following criteria: (1) morphology, (2) esterase isozyme profiles, and (3) the presence of nopaline in leaf extracts. All of the plants regenerated were morphologically more similar to N. tabacum than to N. plumbaginifolia, and many were indistinguishable from N. tabacum. It was found that 37 plants displayed one or two esterases characteristic of N. plumbaginifolia in addition to a full set of esterases from N. tabacum. Based on their esterases, we have classified these plants as somatic hybrids. However, irradiation has clearly reduced the amount of N. plumbaginifolia genetic material that they retain; 24 plants were found that had only N. tabacum esterases but that produced nopaline and were kanamycin resistant. Genomic DNA from several of these plants was probed by Southern blotting for the presence of the authentic neomycin phosphotransferase gene (kanamycin-resistance gene) — all were found to contain the gene. These plants were classified as asymmetric hybrids. Finally, 25 plants were regenerated that were kanamycin sensitive, negative for nopaline, and contained only N. tabacum esterases. All of the regenerated plants, including this final category, were male sterile. As transferring the N. plumbaginifolia cytoplasm to an N. tabacum nuclear background results in an alloplasmic form of male sterility, all of the plants regenerated in this study appear to be cybrids irrespective of their nuclear constitution. Chromosome analysis of the asymmetric hybrids showed that most of them contained one more chromosome than is normal for N. tabacum. The somatic hybrids examined all had several additional chromosomes. Although male sterile, the asymmetric hybrids were female fertile to varying degrees and were successfully backcrossed with N. tabacum. Analysis of the resultant F₁ progeny indicated that the kanamycin-resistance gene from N. plumbaginifolia is partially unstable during meiosis, as would be expected for factors inherited on an unpaired chromosome.Abbreviations Km ^r kanamycin resistant - Km ^s kamacysin sensitive - Nop ⁺ nopaline positive - Nop ^– nopaline negative     

Intergeneric asymmetric hybrids between Nicotiana plumbaginifolia and Atropa belladonna obtained by “gamma-fusion”

Summary Asymmetric nuclear hybrids have been obtained by fusion of cells from a nitrate-reductase deficient mutant of Nicotiana plumbaginifolia (cnx20) and gamma irradiated protoplasts of Atropa belladonna (irradiation doses tested were 10, 30, 50 and 100 krad). The hybrid formation frequency following selection for genotypic complementation in the NR function was in the range of 0.7%–3.7%. Cytogenetic studies demonstrated that all hybrid plants tested possessed multiple (generally tetra- or hexaploid) sets of N. plumbaginifolia (n = 10) chromosomes along with 6–29 Atropa chromosomes (n = 36), some of which were greatly deleted. Besides the cnxA gene (the selection marker), additional material of the irradiated partner was expressed in some of the lines, as shown by analyses of multiple molecular forms of enzymes. Surprisingly, rDNA genes of both parental species were present and amplified in the majority of the hybrids. Whenever studied, the chloroplast DNA in the hybrids was derived from the Nicotiana parent. Regenerants from some lines flowered and were partially fertile. It is concluded that irradiation of cells of the donor parent before fusion can be used to produce highly asymmetric nuclear hybrid plants, although within the dose range tested, the treatment determined the direction of the elimination but not the degree of elimination of the irradiated genome.Abbreviations and Definitions Cp chloroplast - CsCl/EtBr cesiumchloride/ethidiumbromide - EDTA ethyldiamine tris acetate - NR nitrate reductase - SDS sodiumdodecylsulphate - Gamma-fusion is a conventional abbreviation for fusion experiments in which one fusion partner (the donor) is inactivated by sub- or lethal doses of irradiation before fusion with the other (the recipient) partner.     

Chloroplast transfer in Nicotiana based on metabolic complementation between irradiated and iodoacetate treated protoplasts

Protoplasts of a light sensitive plastome mutant of Nicotiana tabacum (2 n=48) were irradiated and fused with iodoacetate-treated Nicotiana plumbaginifolia (2 n=20) protoplasts. Treated parental protoplasts were unable to divide. Metabolic complementation, however, helped the recovery of interspecific fusion products which survived and formed calli. Altogether 40 clones were investigated. N. plumbaginifolia plants were obtained in 15 clones (38%), somatic hybrids in 23 clones, and both types of regenerates were found in 2 clones. Irradiation therefore significantly increased the frequency of segregant formation with the non-irradiated N. plumbaginifolia nuclei (the frequency was 1.4% in the absence of irradiation). Regenerated plants in most cases (31 out of 34) contained chloroplasts from the irradiated parent. In 6 clones plants were obtained with both types of chloroplast. Thus, irradiated N. tabacum chloroplasts had an improved chance of dominating the heterokaryonderived cells, many of which contained N. plumbaginifolia nucleus. The system described should be generally applicable for the transfer of chloroplasts without the use of selectable genetic markers.     

Hybrid genes in the analysis of transformation conditions

Direct gene transfer into plant protoplasts has been recently developed, and conditions for high frequency transformation of SR₁ tobacco protoplasts established. In this paper we analyse numerous transformation parameters in a comparative study on SR₁ Nicotiana tabacum and N. plumbaginifolia, and report on a simple chemical technique for very efficient protoplast transformation. It is based on the synergistic interaction of MgCl₂ and PEG. The technique yielded up to 1400 transformants per 3×10⁵ treated N. tabacum protoplasts (up to 4.8% of the survivors, late selected clones). Using N. plumbaginifolia, the frequencies were 10-fold lower, indicating that the competence for transformation has a species-specific component.Abbreviations PEG polyethyleneglycol - RTF relative transformation frequency - ATF absolute transformation frequency     

Asymmetric hybridization in Nicotiana by “gamma fusion” and progeny analysis of self-fertile hybrids

Summary Mesophyll protoplasts of the nitrate-reductase (NR)-deficient Nicotiana plumbaginifolia mutant, Nia26, were fused with -irradiated mesophyll protoplasts of Nicotiana sylvestris, V-42. Hybrid selection was based on complementation of NR deficiency by transfer of the donor NR gene to N. plumbaginifolia. Regenerated hybrids had different numbers of donor chromosomes in a tetraploid background of N. plumbaginifolia. The transfer and expression of different isozymes from the donor were also observed. Six self-fertile regenerants were obtained from 21 independently isolated cell colonies. Progeny analyses revealed: (1) the linkage of NR and shikimate dehydrogenase (ShDh); (2) a stabilization of the transmission rate of NR; and (3) the obtainment of mono- and disomic addition lines in the first and second progeny of the original regenerants. Southern hybridization analyses demonstrated unequivocally the presence of the NR gene from the donor partner in progeny plants.     

A light sensitive recipient for the effective transfer of chloroplast and mitochondrial traits by protoplast fusion in Nicotiana

Summary A light sensitive mutant was used as a recipient in the transfer of chloroplasts from a wildtype donor. Gamma irradiated (lethal dose) mesophyll protoplasts of Nicotiana gossei were fused with mesophyll protoplasts of a N. plumbaginifolia line carrying light sensitive plastids from a N. tabacum mutant. After fusion, colonies containing wild-type plastids from the cytoplasm donor were selected by their green colour. Most of the regenerated plants had N. plumbaginifolia morphology, but were a normal green in colour. The presence of donor-type plastids was confirmed by the restriction pattern of chloroplast DNA in each plant analysed. These cybrids were fully male sterile with an altered flower morphology typical of certain types of alloplasmic male sterility in Nicotiana. The use of the cytoplasmic light sensitive recipient proved to be suitable for effective interspecific transfer of wild-type chloroplasts. The recombinant-type mitochondrial DNA restriction patterns and the male sterility of the cybrids indicated the co-transfer of chloroplast and mitochondrial traits. On leave from: Department of Genetics, Section of Biosciences, Martin Luther University, Domplatz 1, DDR-4020 Halle/ S., German Democratic Republic     

Detectable ,-glucuronidase activity in transgenic cranberry is affected by endogenous inhibitors and plant development

Extracts of cranberry, rich in flavonols and proanthocyanins, inactivated-glucuronidase (GUS) in assays using either purified bacterial GUS or preparations of transgenic tobacco (Nicotiana tabacum L.) or transgenic cranberry (Vaccinium macrocarpon Ait.) expressing thegusA gene. Histochemical GUS assays produced random and generally unpredictable staining. The addition of poly-vinylpolypyrrolidone (PVPP) during the preparation of transgenic cranberry leaf extracts increased the detectable GUS activity in fluorogenic assays more than 200-fold. Detectable GUS activity varied among transclones and also within a transclone depending on the developmental and physiological state of the tissue, as well as the growth environment. Use of PVPP altered the relative ranking of plants based on their total transgenic enzyme activity and resulted in different conclusions as to the effects of genotype or growth environment on transgene expression.Abbreviations GUS -Glucuronidase - MUG 4-Methyl umbelliferyl glucuronide - PVPP Polyvinylpolypyrrolidone - X-gluc 5-Bromo-4-chloro-3-indolyl glucuronide     

Silicon carbide fiber-mediated DNA delivery into plant cells

Summary Silicon carbide fiber-mediated delivery of DNA into intact plant cells was investigated. Black Mexican Sweet (BMS) maize (Zea mays) and tobacco (Nicotiana tabacum) suspension culture cells were vortexed in the presence of liquid medium, plasmid DNA encoding -glucuronidase (GUS), and silicon carbide fibers. Penetration of BMS cells by the silicon carbide fibers was observed by scanning electron microscopy of vortexed cells. Following fiber and DNA treatment, BMS cells transiently expressed GUS activity at a mean frequency of 139.5 units (one unit = one blue cell or one colony of blue cells) per sample. Treated tobacco cells expressed an average of 373 GUS units per sample. Untreated controls did not exhibit GUS activity. These results indicate that the silicon carbide fibers-vortex procedure can be used to rapidly and inexpensively deliver foreign DNA into intact plant cells for investigations of transient gene expression.Abbreviations BMS Black Mexican Sweet maize suspension cultures - MS Murashige and Skoog salts - GUS -glucuronidase - 2,4-D 2,4 dichlorophenoxyacetic acid     

Cytoplast-protoplast fusion for interspecific chloroplast transfer in Nicotiana

Summary Protoplasts of Nicotiana tabacum (SR1), carrying a maternally-inherited streptomycin resistance mutation, were enucleated by centrifugation through a Percoll gradient. The resulting cytoplasts containing resistant plastids, were fused with sensitive Nicotiana plumbaginifolia protoplasts. The SR1 cytoplasts, having no nuclei, were unable to form calli. All resistant clones recovered after fusion-induction were therefore supposed to be derived from interspecific cytoplast-protoplast fusion. N. plumbaginifolia plants regenerated in 17 out of the 75 resistant clones studied. Plants obtained from eight of these clones were resistant to streptomycin and inherited the resistance maternally, as expected when transferring SR1 plastids into the N. plumbaginifolia nuclear background. Plastid transfer in these plants has been confirmed by the EcoRI restriction pattern of the chloroplast DNA.In nine clones N. plumbaginifolia plants were sensitive although obtained from initially resistant clones. This phenomenon is explained by the maintenance of plastid heterogeneity on the selective streptomycin medium, and formation of plants from sensitive sectors on the non-selective regeneration medium.SR1 protoplasts, originally present as contaminants in the cytoplast preparation (2–7%) did not form colonies (or very rarely) after polyethylene glycol treatment. The nuclei from such protoplasts were recovered, however, in the interspecific somatic hybrids (56 clones), and in segregants having the SR1 nucleus but some cytoplasm from N. plumbaginifolia (2 clones). The majority (about 80%) of the recovered resistant clones therefore acquired the streptomycin resistance factor from the rare (2–7%) contaminating SR1 protoplasts. This is explained by the protoplasts being more stable during fusion induction.     

Chromosomal analysis of Nicotiana asymmetric somatic hybrids by dot blotting and in situ hybridization

Summary A species-specific, dispersed repetitive DNA sequence was cloned from Nicotiana plumbaginifolia and used in dot blots and in situ hybridizations to analyze asymmetric somatic hybrids of N. tabacum(+)kanamycin-resistant N. plumbaginifolia. Dot blot hybridization data, using the cloned, species-specific repetitive DNA as a probe, showed that some of the hybrids contain only 1%–5% N. plumbaginifolia DNA, whereas others contain 15%–25%. In situ hybridization of the probe to chromosome spreads showed that the extremely asymmetric hybrids retain a single N. plumbaginifolia chromosome; the hybrids with higher dot blot values were found to have 8 to 12 N. plumbaginifolia chromosomes and chromosome fragments. In situ hybridization also revealed translocations between N. plumbaginifolia and N. tabacum chromosomes in 3 of 8 hybrids studied. RFLP analysis using a 5S gene probe showed the presence of N. plumbaginifolia-specific 5S banding patterns in most hybrids examined, including those that retain only a single N. plumbaginifolia chromosome.     

Transfer of kanamycin resistance fromNicotiana tabacum toNicotiana plumbaginifolia by fusion of x-irradiated protoplasts

Summary Nuclear hybrids have been obtained by fusion of mesophyll protoplasts ofNicotiana plumbaginifolia and x-irradiated or iodoacetate-treated mesophyll protoplasts of a kanamycin-resistant line ofN. tabacum. The effect of irradiation on the recovery of asymmetric hybrids was evaluated by analysis of their morphology, fertility, chromosome number, isozyme patterns, restriction patterns in their organelle DNAs, and presence of the kanamycin-resistance gene. The results presented in this paper show that x-ray irradiation leads to a significant reduction in the amount ofN. tabacum genome present in the hybrids and demonstrates, once more, the power of this technique to induce directional loss of genomic traits of the irradiated parent.     

An S-RNase promoter from Nicotiana alata functions in transgenic N. alata plants but not Nicotiana tabacum

Nicotiana tabacum and Nicotiana alata plants were transformed with genomic clones of two S-RNase alleles from N. alata. Neither the S ₂ clone, with 1.6 kb of 5 sequence, nor the S ₆ clone, with 2.8 kb of 5 sequence, were expressed at detectable levels in transgenic N. tabacum plants. In N. alata, expression of the S ₂ clone was not detected, however the S ₆ clone was expressed (at low levels) in three out of four transgenic plants. An S ₆-promoter-GUS fusion gene was also expressed in transgenic N. alata but not N. tabacum. Although endogenous S-RNase genes are expressed exclusively in floral pistils, the GUS fusion was expressed in both styles and leaves.     

Molecular genetic characterization of plant somatic hybrids

An efficient and easy method for genetic characterization of plant somatic hybrids is proposed. In a first qualitative approach, four somatic hybrids and their parental species (Nicotiana tabacum andN. plumbaginifolia) were characterized by DNA fingerprinting and Random Amplification of Polymorphic DNA (RAPD). After this, a quantitative estimation of the degree of parental contribution to the hybrids was carried out by means of a slot-blot analysis. Both qualitative methods, showed one hybrid identical toN. tabacum, two almost identical toN. plumbaginifolia, and a fourth similar to this parental species, but with someN. tabacum admixture. The quantitative method, for the same hybrids, gave 83%, 7%, 7%, and 37%N. tabacum DNA contribution, respectively.     

Culture of plant somatic hybrids following electrical fusion

Summary Electrically-induced protoplast fusion has been used to produce somatic hybrids between Nicotiana plumbaginifolia and Nicotiana tabacum. Following fusion of suspension culture protoplasts (N. plumbaginifolia) with mesophyll protoplasts (N. tabacum) heterokaryons were identified visually and their development was followed in culture. Because electrical fusion is a microtechnique, procedures were developed for culturing the heterokaryons in small numbers and at low density. The fusion and culture procedures described are rapid, uncomplicated and repeatable. Good cell viabilities indicate that the fusion procedure is not cytotoxic. Fused material was cultured 1–2 days at high density in modified K3 medium (Nagy and Maliga 1976). The heterokaryons were isolated manually and grown, at low density in conditioned media. Calli have been regenerated. Esterase isozyme patterns confirm the hybrid character of calli and clonally-derived plantlets recovered from these fusions.