Large scale isolation of maternally inherited lincomycin resistance mutations, in diploid Nicotiana plumbaginifolia protoplast cultures

Summary Lincomycin-resistant clones were isolated in diploid protoplast cultures of Nicotiana plumbaginifolia. Selection of the resistant clones was based on the ability of resistant calli to green in the presence of the antibiotic (1,000 mg l^-1). Sensitive colonies formed white calli under the same conditions. In the absence of mutagenic treatment the frequency of the resistant clones was 1.0×10^-4. This frequency could be increased up to 5.8×10^-4 and 7.2×10^-4 by treatment with 0.1 mM and 0.3 mM N-ethyl-N-nitrosourea (NEU), respectively.Regenerated plants of 56 clones were tested for lincomycin resistance. Regenerates from all but seven clones were resistant to lincomycin, as demonstrated by leaf assay. The lincomycin-resistant regenerates tested were also resistant to clindamycin (a lincomycin derivative), but sensitive to streptomycin.Regenerated plants in 17 clones were fully fertile and inherited lincomycin resistance maternally. Segregation for lincomycin resistance was observed in the seed progeny of five clones, which indicated maintenance of mixed cytoplasmic determinants after plant regeneration. Seed transmission of lincomycin resistance was confirmed in an additional 17 clones but the mode of inheritance (maternal or Mendelian) was not determined because of pollen sterility or reduced seed germination ability. These defects first appeared when the higher concentration of NEU was used. Various pigment deficiencies were also observed in a few clones.     

Amino acid auxotrophs from protoplast cultures of Nicotiana plumbaginifolia, Viviani

Summary Several amino acid requiring auxotrophs have been isolated from unsupplemented protoplast cultures of haploid Nicotiana plumbaginifolia following incubation with BUdR (1-5x10^-5, 2 days) and recovery on complete medium. The auxotrophic lines required the following amino acid(s) for growth: his, ile, leu, ile+val, met or try. Met^– is a new type isolated in higher plants. The same absolute amino acid requirement was observed in plants regenerated from auxotrophic cultures. Precursor feeding tests, enzyme assays, and/or metabolic complementation through protoplast fusion were used to identify the genetic lesion leading to auxotrophy. Mutant seeds were obtained from supplemented Met^– plants. Seeds were also collected from selfed plants regenerated from various complementing fusion products, and a His^– revertant. Genetic analysis indicated that under natural conditions of seed formation amino acid auxotrophy-in contrast to NR deficiency-failed to segregate in progeny tests.Abbreviations and definitions BUdR and FUdR 5-bromo- and fluoro-deoxyuridine respectively - AP imidazole acetol phosphate - IGP imidazole glycerol phosphate - NR nitrate reductase - NAA naphthaleneacetic acid - BAP 6-benzylaminopurine - TIP total isolation procedure - ER Escape rate—the proportion of the selected cell population surviving the BUdR treatment - BR Recovery rate—the proportion of clones identified as amino acid auxotrophs from total escaping clones - TS Total surviving colonies—the number of inoculated protoplasts/variant x plating efficiency - TST Total starvation time—the number of days on minimal medium (preincubation time+BUdR incubation time). The relationship days vs. number of divisions is as follows: 3- to 4-day-old protoplasts, 1 division; 5–6 days, 2 divisions; 7–8 days, 3 divisions Dedicated to Professor Georg Melchers to celebrate his 50-year association with the journal     

Mutants of Nicotiana plumbaginifolia with specific resistance to auxin

Summary We have isolated nine independent auxin-resistant mutants of Nicotiana plumbaginifolia by culturing M₂ seedlings in the presence of indole-3-acetic acid ethyl ester or 1-naphthaleneacetic acid at concentrations which significantly inhibit hypocotyl elongation of the wild type. The mutations were induced by treating seed with ethyl methanesulphonate and were found in the course of screening 10 000 individual M₂ families. Auxin resistance was in all cases the result of a mutation at a single, nuclear locus. The dominance relationships of two of the mutants could be defined as recessive or dominant; all other mutants showed partial dominance. In contrast to previously described mutants of Arabidopsis and N. plumbaginifolia, all of the present mutants were specifically resistant to auxin; the mutants were cross-resistant to several auxins, but showed no increased resistance to cytokinin, abscisic acid, ethylene or 1-amino-cyclopropane-1-carboxylic acid. The importance of the choice of the selection criterion for the isolation of specific resistance traits is discussed.     

Subcellular location of lincomycin resistance in Nicotiana mutants

Summary Lincomycin-resistant Nicotiana plumbaginifolia plastid mutants were considered also to carry mitochondrial mutations on the basis of their ability to grow in the dark under selective conditions. To clarify the role of mitochondria, individual protoplasts of the green, lincomycin-resistant N. plumbaginifolia mutant LR400 were microfused with protoplasts of the N. tabacum plastid albino line 92V37, which possesses N. undulata cytoplasm. The production of lincomycin-resistant albino cybrid lines, with N. undulata plastids and recombinant mitochondria, strongly indicated a determining role for mitochondria in the lincomycin resistance. Sequence analysis of the region encompassing putative mutation sites in the 26S rRNA genes from the LR400 and several other lincomycin-resistant N. plumbaginifolia mutants revelaed, however, no differences from the wild-type sequence. As an alternative source of the resistance of the fusion products, the N. tabacum fusion partner was also taken into account. Surprisingly, a natural lincomycin resistance of tobacco was detected, which was inherited as a dominant nuclear trait. This result compromises the interpretation of the fusion data suggested above. Thus, to answer the original question definitively, the mutant LR400 was crossed as a female parent with a N. plumbaginifolia line carrying streptomycin-resistant N. tabacum plastids. Calli were then induced from the seedlings. Occasional paternal plastid transmissions were selected as streptomycin-resistant calli on selective medium. These cell lines were shown by restriction enzyme analysis to contain paternal plastids and maternal mitochondria. They were tested for greening and growing ability in the presence of lincomycin. These resistance traits proved to be genetically linked and exclusively located in the plastids.EMBL accession number X68710     

Transposition of the maize autonomous element Activator in transgenic Nicotiana plumbaginifolia plants

The maize autonomous transposable element Ac was introduced into haploid Nicotiana plumbaginifolia via Agrobacterium tumefaciens transformation of leaf disks. All the regenerated transformants (R0) were diploid and either homozygous or heterozygous for the hygromycin resistance gene used to select primary transformants. The Ac excision frequency was determined using the phenotypic assay of restoration of neomycin phosphotransferase activity and expression of kanamycin resistance among progeny seedlings. Some of the R0 plants segregated kanamycin-resistant seedlings in selfed progeny at a high frequency (34 to 100%) and contained one or more transposed Ac elements. In the primary transformants Ac transposition probably occurred during plant regeneration or early development. Other R0 transformants segregated kanamycin-resistant plants at a low frequency ( 4%). Two transformants of this latter class, containing a unique unexcised Ac element, were chosen for further study in the expectation that their kanamycin resistant progeny would result from independent germinal transposition events. Southern blot analysis of 32 kanamycin-resistant plants (R1 or R2), selected after respectively one or two selfings of these primary transformants, showed that 27 had a transposed Ac at a new location and 5 did not have any Ac element. Transposed Ac copy number varied from one to six and almost all transposition events were independent. Southern analysis of the R2 and R3 progeny of these kanamycin-resistant plants showed that Ac continued to transpose during four generations, and its activity increased with its copy number. The frequency of Ac transposition, from different loci, remained low ( 7%) from R0 to R3 generations when only one Ac copy was present. The strategy of choosing R0 plants that undergo a low frequency of germinal excision will provide a means to avoid screening non-independent transpositions and increase the efficiency of transposon tagging.     

Intertrubal chloroplast transfer by protoplast fusion between Nicotiana tabacum and Salpiglossis sinuata

Summary Chloroplast tranfer was achieved by protoplast fusion between Nicotiana tobacum (Cestreae, Cestroideae) and Salpiglossis sinuata (Salpiglossideae, Cestroideae) in the family Solanaceae. Isolation of cybrid clones was facilitated by irradiation of the cytoplasm donor protoplasts, and the use of appropriate plastid mutants, streptomycin-resistant as donor, or light-sensitive as recipient. Cybrid colonies were selected by their green colour against the background of bleached (light-sensitive or streptomycin-sensitive) colonies. In the Nicotiana (Salpiglossis) cybrid plants possessing normal tobacco morphology and chromsome number, the presence of Salpiglossis, plastids was verified by restriction analysis of the chloroplast DNA. A similar analysis of the mitochondrial DNA of these lines revealed unique, recombinant patterns in the case of both fertile and sterile plants. Progeny showed no appearance of chlorophyll-deficiency in F₁ and an additional back-cross generation. Attempts at transfer of entire chloroplasts between Nicotiana tabacum and Solanum nigrum (Solaneae, Solanoideae) did not result in any cybrid cell lines in a medium suitable for green colony formation of both species. These results suggest that fusion-mediated chloroplast transfer can surmount a considerable taxonomical distance, but might be hampered by a plastome-genome incompatibility in more remote combinations.     

Interspecific hybridization between Penicillium chrysogenum and Penicillium cyaneo-fulvum following protoplast fusion

Summary Slow-growing interspecific heterokaryons were isolated on minimal medium following the induced fusion of protoplasts from auxotrophic mutants of Penicillium chrysogenum and Penicillium cyaneo-fulvum. After 5–7 days cultivation the heterokaryons produced vigorously growing sectors which on transfer gave genetically stable colonies. Cultivation of these colonies on a complete medium supplemented with p-fluorophenylalanine or benomyl broke down this stability and several different prototrophic and auxotrophic colony types were isolated. Many of these behaved as diploids or aneuploids showing sectoring either spontaneously, or in the presence of an haploidizing agent. Some of the latter isolates were recombinants for parental spore colour and auxotrophic markers.     

The use of cytoplasmic streptomycin resistance: Chloroplast transfer from Nicotiana tabacum into Nicotiana sylvestris, and Isolation of their somatic hybrids

Summary Leaf protoplasts of Nicotiana tabacum SR1 (2n=4x=48) treated with iodoacetate (10 mM; 25 C; 30 min) and consequently unable to divide, and untreated leaf protoplasts of Nicotiana sylvestris (2n=2x=24) were fused using polyethylene glycol (PEG). The SR1 line is resistant to streptomycin because of a maternally inherited mutation, and has streptomycin-insensitive chloroplast ribosomes.After 1 month of growth in the absence of streptomycin protoplast-derived calli were plated into selective medium (1,000 g ml^-1 streptomycin) and the resistant clones were isolated. Out of 10⁶ PEG-treated protoplasts (1:1 mixture of parental types) 137 resistant (green) clones were obtained, whereas in the same number of parental cells, not subjected to fusion induction, no resistant callus was found.At least four plants were regenerated from each of the clones. The regenerates were identified as somatic hybrids (H), N. sylvestris (Ns) or N. tabacum (Nt) by looking at esterase and peroxidase isoenzymes and morphology. The three types of regenerates were distributed amongst the clones as follows: H only (105 clones); Ns (16 clones); Ns+H (6 clones); Nt only (3 clones); Nt+H (6 clones); Nt+Ns (1 clone). The high proportion of hybrid regenerates indicates that nuclear fusion has occured in the overwhelming majority of the heterokaryocytes. Cytoplasmic mutations in combination with inactivation by iodoacetate, therefore, are suitable markers to produce somatic hybrids. Segregation of nuclei after fusion resulted in new combinations of organelles and nuclei, the final outcome being the transfer of resistant chloroplasts into N. sylvestris, some of which have the original diploid (2n=24) chromosome number. Data suggest that segregants were in most cases obtained from multiple fusions. Streptomycin resistance was inherited maternally in the N. sylvestris (six clones) tested and the hybrid (three clones) regenerates.     

Immunological evidence for transfer of the barley nitrate reductase structural gene to Nicotiana tabacum by protoplast fusion

Summary A protoplast fusion experiment was designed in which the selectable marker, nitrate reductase (NR), also served as a biochemical marker to provide direct evidence for intergeneric specific gene transfer. NR-deficient tobacco (Nicotiana tabacum) mutant Nia30 protoplasts were the recipients for the attempted transfer of the NR structural gene from 50 krad -irradiated barley (Hordeum vulgare L.) protoplasts. Barley protoplasts did not form colonies and Nia30 protoplasts could not grow on nitrate medium; therefore, selection was for correction of NR deficiency allowing tobacco colonies to grow on nitrate medium. Colonies were selected from protoplast fusion treatments at an approximate frequency of 10^-5. This frequency was similar to the Nia30 reversion frequency, and thus provided little evidence for transfer of the barley NR gene to tobacco. Plants regenerated from colonies had NR activity and were analyzed by western blotting using barley NR antiserum to determine the characteristics of the NR conferring growth on nitrate. Ten plants exhibited tobacco NR indicating reversion of a Nia30 mutant NR locus. Twelve of 26 regenerated tobacco plants analyzed had NR subunits with the electrophoretic mobility and antigenic properties of barley NR. These included plants regenerated from colonies selected from 1) co-culturing a mixture of Nia30 protoplasts with irradiated barley protoplasts without a fusion treatment, 2) a protoplast fusion treatment of Nia30 and barley protoplasts, and 3) a fusion treatment of Nia30 protoplasts with irradiated barley protoplasts. No barley-like NR was detected in plants regenerated from a colony that grew on nitrate following selfed fusion of Nia30 protoplasts. Because tobacco plants expressing barley-like NR were recovered from mixture controls as well as fusion treatments, explanations for these results other than protoplast fusionmediated gene transfer are discussed.     

Selection of a Nicotiana plumbaginifolia universal hybridizer and its use in intergeneric somatic hybrid formation

Summary A Nicotiana plumbaginifolia cell strain carrying a positive (dominant) trait, resistance to azetidine-2-carboxylate (A2C), was selected in strain NX1 which lacked nitrate reductase activity (a negative or recessive trait). This universal hybridizer strain, denoted NXA^r, was fused with dextran to a Daucus carota strain, PR, which carried glyphosate (GLP) resistance. A large number of hybrids were selected in a medium with NO ₃ ^- as the sole nitrogen source and A2C as inhibitor, conditions which prevent the growth of both parents. When the selected colonies were then tested for GLP resistance, 93% carried this trait. In addition the hybrid nature was indicated by additive chromosome numbers, both A2C and GLP resistance in suspension cultures, intermediate nitrate reductase activity and the presence of banding patterns for three isozymes which match those of the parents. Southern hybridization analysis using an enolpyruvylshikimic acid-3-phosphate synthase (EPSPS) probe, pMON 6145, also showed the presence of the gene from both parents in the hybrid strains based on restriction length polymorphisms. The PR strain contains increased levels of EPSPS which confers GLP^r due to gene amplification. Since the universal hybridizer can be used as a fusion partner with any wild-type line many protoplast fusion studies can be carried out easily.Abbreviations A2C azetidine-2-carboxylate - 2,4-D 2,4-dichlorophenoxyacetic acid - EPSPS 5-enolpyruvylshikimic acid-3-phosphate synthase - GLP glyphosate - HAT hypoxanthine, aminopterin, glycine and thymidine medium - IDH isocitrate dehydrogenase - MDH malate dehydrogenase - 5MT 5-methyltryptophan - NBT nitroblue tetrazolium - PGI phosphoglucoisomerase - SDS sodium dodecylsulfate     

Protoplast fusion mediated transfer of oligomycin resistance from Nicotiana sylvestris to Solanum tuberosum by intergeneric cybridization

Summary We have successfully bridged the intergeneric barriers between Nicotiana and Solanum with respect to chondriome transfer. To enable this transfer we utilized the donor-recipient protoplast-fusion procedure. Consequently protoplasts of a Nicotiana sylvestris line with putativly oligomycin-resistant mitochondria (line Oli ^R 38) were used as irradiated chondriome donors and iodoacetate-treated protoplasts of Solanum tuberosum cv. Desiree served as recipients. The plated fusion products as well as their derived colonies and calli were exposed to gradually increasing levels of oligomycin. The resulting plantlets had potato morphology and were analyzed with respect to their mitochondrial DNA and chloroplast DNA. Fifteen out of 50 regenerated plants were verified as true cybrids. Detailed analyses of one cybrid revealed chondriome components from the oligomycin-resistant donor line, Oli ^R 38, but retention of the plastome of potato. This cybrid was oligomycin-resistant as revealed by root-culture analysis. It was thus verified that due to selection, chondriome components could be transferred from a N. sylvestris donor into a cybrid having all the phenotypic features controlled by the nucleus of the recipient fusion partner (S. tuberosum).     

Isolation and molecular characterization of dTnp1, a mobile and defective transposable element of Nicotiana plumbaginifolia

By Northern blot analysis of nitrate reductase-deficient mutants of Nicotiana plumbaginifolia, we identified a mutant (mutant D65), obtained after -ray irradiation of protoplasts, which contained an insertion sequence in the nitrate reductase (NR) mRNA. This insertion sequence was localized by polymerase chain reaction (PCR) in the first exon of NR and was also shown to be present in the NR gene. The mutant gene contained a 565 by insertion sequence that exhibits the sequence characteristics of a transposable element, which was thus named dTnp1. The dTnp1 element has 14 by terminal inverted repeats and is flanked by an 8-bp target site duplication generated upon transposition. These inverted repeats have significant sequence homology with those of other transposable elements. Judging by its size and the absence of a long open reading frame, dTnp1 appears to represent a defective, although mobile, transposable element. The octamer motif TTTAGGCC was found several times in direct orientation near the 5 and 3 ends of dTnp1 together with a perfect palindrome located after the 5 inverted repeat. Southern blot analysis using an internal probe of dTnp1 suggested that this element occurs as a single copy in the genome of N. plumbaginifolia. It is also present in N. tabacum, but absent in tomato or petunia. The dTnp1 element is therefore of potential use for gene tagging in Nicotiana species.     

Somatic transfer of cytoplasmic traits in Brassica napus L. by haploid protoplast fusion

Summary Fusions between protoplasts from haploid cytoplasmic atrazine resistant (CATR) and haploid cytoplasmic male sterile (CMS) Brassica napus plants were used to produce a diploid CMS/CATR cybrid. The hybrid nature of the cytoplasm was confirmed by comparing the EcoRI restriction fragment patterns of chloroplast and mitochondrial DNA from the cybrid with the parental patterns. The advantages of using haploid protoplasts for fusion experiments as well as the utilization of the CMS/CATR cybrid for hybrid seed production are discussed.     

Marker rescue from the Nicotiana tabacum plastid genome using a plastid/Escherichia coli shuttle vector

We recently reported an 868-bp plastid DNA minicircle, NICE1, that formed during transformation in a transplastomic Nicotiana tabacum line. Shuttle plasmids containing NICEI sequences were maintained extrachromosomally in plastids and shown to undergo recombination with NICE1 sequences on the plastid genome. To prove the general utility of the shuttle plasmids, we tested whether plastid genes outside the NICE1 region could be rescued in Escherichia coli. The NICE1-based rescue plasmid, pNICER1, carries NICE1 sequences for maintenance in plastids, the CoIE1 ori for maintenance in E. coli and a spectinomcyin resistance gene (aadA) for selection in both systems. In addition, pNICERl carries a defective kanamycin resistance gene, kan*, to target the rescue of a functional kanamycin resistance gene, kan, from the recipient plastid genome. pNICERl was introduced into plastids where recombination could occur between the homologous kan/kan* sequences, and subsequently rescued in E. coli to recover the products of recombination. Based on the expression of kanamycin resistance in E. coli and the analysis of three restriction fragment polymorphisms, recombinant kan genes were recovered at a high frequency. Efficient rescue of kan from the plastid genome in E. coli indicates that NICE 1-based plasmids are suitable for rescuing mutations from any part of the plastid genome, expanding the repertoire of genetic tools available for plastid biology.     

Interspecific protoplast fusion for the transfer of atrazine resistance from Solanum nigrum to tomato (Lycopersicon esculentum. L)

TAES Technical Article #23184     

A cell line of Nicotiana sylvestris with resistance to kanamycin and streptomycin

Summary Cell lines resistant to 50 g ml^-1 kanamycin sulphate were isolated from cell suspension cultures initiated from a haploid Nicotiana sylvestris plant. One line, KR103, has been studied in detail. Resistance of this line was shown to be stable in the absence of the drug. KR103 was found also to be resistant to streptomycin, another inhibitor of 70S ribosomal protein synthesis.Both KR103 and the sensitive line convert kanamycin, but not streptomycin, to a form which is no longer effective in a bacterial bioassay, while maintaining its toxicity for sensitive plant cells.KR103 is defective in morphogenesis and plastid development.     

Transfer of resistance to Xanthomonas campestris pv campestris into Brassica oleracea L. by protoplast fusion

Black rot caused by the bacterium Xanthomonas campestris pv campestris is one of the most serious diseases of Brassica oleracea. Since sources of resistance to the disease within B. oleracea are insufficient and control means are limited, the development of resistant breeding lines is extremely desirable. Certain lines of B. napus contain very high resistance controlled by a dominant gene, but crossing the two species sexually is very difficult. Therefore, somatic hybrids were produced by protoplast fusion between rapid cycling B. oleracea and a B. napus line highly resistant to X. campestris pv campestris. Hybrid identity was confirmed by morphological studies, flow cytometric estimation of nuclear DNA content, and analysis of random amplified polymorphic DNA (RAPD). Inoculations with the pathogen identified four somatic hybrids with high resistance. The resistant hybrid plants were fertile and set seed when selfed or crossed reciprocally to the bridge line 15 (Quazi 1988). Direct crosses to B. oleracea were unsuccessful, but embryo rescue facilitated the production of a first-backcross generation. The BC₁ plants were resistant to the pathogen. Progeny from the crosses to line 15 were all susceptible. Embryo rescue techniques were not obligatory for the development of a second-backcross generation, and several resistant BC₂ plants were obtained.