Transformation of Montmorency sour cherry (Prunus cerasus L.) and Gisela 6 (P. cerasus x P. canescens) cherry rootstock mediated by Agrobacterium tumefaciens.

Sour cherry (Prunus cerasus L.) scion cv. Montmorency and rootstock cv. Gisela 6 (P. cerasus x P. canescens) were transformed using Agrobacterium tumefaciens strain EHA105:pBISN1 carrying the neomycin phosphotransferase gene (nptII) and an intron interrupted ss-glucuronidase (GUS) reporter gene (gusA). Whole leaf explants were co-cultivated with A. tumefaciens, and selection and regeneration of transformed cells and shoots of both cultivars was carried out for 12 weeks on selection medium containing 50 mg l(-1) kanamycin (Km) and 250 mg l(-1) timentin. These media were [Quoirin and Lepoivre (Acta Hortic 78:437-442, 1977)] supplemented with 0.5 mg l(-1) benzylaminopurine (BA) + 0.05 mg l(-1) indole-3-butyric acid (IBA), and woody plant medium [Lloyd and McCown (Proc Int Plant Prop Soc 30:421-427, 1980)] containing 2.0 mg l(-1) BA + 1.0 mg l(-1) IBA for cv. Montmorency and cv. Gisela 6, respectively. Seven out of 226 (3.1%) explants of cv. Montmorency and five out of 152 (3.9%) explants of cv. Gisela 6 produced 30/39 GUS- and PCR-positive shoots from the cut midribs via an intermediate callus. Southern analysis of the GUS- and PCR-positive transformants confirmed stable integration of the transgenes with 1-3 copy numbers in the genomes of seven lines of cv. Montmorency and five of cv. Gisela 6. The selected transformants have a normal phenotype in vitro.

Agrobacterium tumefaciens-mediated transformation of blueberry (Vaccinium corymbosum L.).

Transient expression studies using blueberry leaf explants and monitored by beta-glucuronidase (GUS) assays indicated Agrobacterium tumefaciens strain EHA105 was more effective than LBA4404 or GV3101; and the use of acetosyringone (AS) at 100 microM for inoculation and 6 days co-cultivation was optimum compared to 2, 4, 8, 10 or 12 days. Subsequently, explants of the cultivars Aurora, Bluecrop, Brigitta, and Legacy were inoculated with strain EHA105 containing the binary vector pBISN1 with the neomycin phosphotransferase gene (nptII) and an intron-interrupted GUS gene directed by the chimeric super promoter (Aocs)3AmasPmas. Co-cultivation was for 6 days on modified woody plant medium (WPM) plus 100 microM AS. Explants were then placed on modified WPM supplemented with 1.0 mg l(-1) thidiazuron, 0.5 mg l(-1) alpha-naphthaleneacetic, 10 mg l(-1) kanamycin (Km), and 250 mg l(-1) cefotaxime. Selection for Km-resistant shoots was carried out in the dark for 2 weeks followed by culture in the light at 30 microE m(-2) s(-1) at 25 degrees C. After 12 weeks, selected shoots that were both Km resistant and GUS positive were obtained from 15.3% of the inoculated leaf explants of cultivar Aurora. Sixty-eight independent clones derived from such shoots all tested positive by the polymerase chain reaction using a nptII primer. Eight of eight among these 68 clones tested positive by Southern hybridization using a gusA gene derived probe. The transformation protocol also yielded Km-resistant, GUS-positive shoots that were also PCR positive at frequencies of 5.0% for Bluecrop, 10.0% for Brigitta and 5.6% for Legacy.

Transformation of the limonene synthase gene into peppermint (Mentha piperita L.) and preliminary studies on the essential oil profiles of single transgenic plants.

Agrobacterium-mediated and direct gene transfer into protoplasts using PEG were both successfully used to produce stable, transformed peppermint plants (Mentha×piperita L. cultivar Black Mitcham) with the limonene synthase gene. Stem internode explants found to possess a high level of organogenesis through adventitious shoot formation were subjected to Agrobacterium tumefaciens disarmed strain GV3101 (pMP90). Following the development of an efficient protoplast-to-plant cycle from stem-isolated protoplasts, they were used in direct gene transformations. In both cases the binary vector pGA643 carrying the nptII/GUS genes, both driven by the CaMV35S promoter, was used in preliminary plant-transformation studies. Later, GUS was replaced with the limonene synthase gene. Kanamycin was used as a selective agent in all transformation experiments to obtain both transformed protoplast-derived calli as well as putative transgenic shoots regenerated from internode explants. Both types of transformation resulted in transgenic plants which were detected using PCR and confirmed by Southern-blot hybridizations. Southern analysis revealed that the method of Agrobacterium-mediated transformation is superior to the direct DNA uptake into protoplasts with regard to the stability of the insert during the transformation event. Single transgenic plants were grown to 10% flowering in a greenhouse and the plants derived both by the Agrobacterium and the protoplast-derived methods were generally observed to have essential oil profiles characterized by a high-menthone, low-menthol, high-menthofuran and -pulegone content in comparison to a typical mid-west peppermint. Limonene varied only slightly, around 1.2%, in transgenic plants produced by both methods.

Combined RAPD and RFLP molecular linkage map of asparagus.

Two linkage maps of asparagus (Asparagus officinalis L.) were constructed using a double pseudotestcross mapping strategy with restriction fragment length polymorphisms (RFLPs), random amplified polymorphic DNAs (RAPDs), and allozymes as markers in a population generated from crossing MW25 x A19, two heterozygous parents. All data were inverted and combined with the natural data to detect linkages in repulsion phase. Two sets of data, one for each parent, were formed according to the inheritance patterns of the markers. The maternal MW25 map has a total of 163 marker loci placed in 13 linkage groups covering 1281 cM, with an average and a maximum distance between adjacent loci of 7.9 and 29 cM, respectively. The paternal A19 map has 183 marker loci covering 1324 cM in 9 linkage groups, with an average and a maximum distance between two adjacent loci of 7.7 and 29 cM, respectively. Six multiallelic RFLPs segregating in the pattern a/c x b/c and eight heterozygous loci (four RAPDs, and four RFLPs segregating in the pattern a/b x a/b (HZ loci)) were common to both maps. These 14 loci were used as bridges to align homologous groups between the two maps. In this case, RFLPs were more frequent and informative than RAPDs. Nine linkage groups in the MW25 map were homologous to six groups in the A19 map. In two cases, two or more bridge loci were common to a group; thus, the orientation of homologous linkage groups was also determined. In four other cases, only one locus was common to the two homologous groups and the orientation was unknown. Mdh, four RFLPs, and 14 RAPDs were assigned to chromosome L5, which also has the sex locus M.

RFLP linkage map of asparagus.

A population resulting from a double pseudotestcross of two outbred-derived asparagus (Asparagus officinalis L.) clones was evaluated by RFLP (restriction fragment length polymorphism) analysis to produce individual maps of the male and female parents. An asparagus PstI genomic library was created and used as the source of probes. Scoring of bands was done by examining SDRFs (single dose restriction fragments) that are present in one parent and absent in the other and segregate 1:1 in the progeny. The data were analyzed as a backcross population; inversion or recoding allowed for the detection of repulsion phase linkage. The male parent map consisted of 33 loci in 10 groups, while the female parent map had 48 loci arranged in 14 groups. Segregation distortion was minimal (5%), and 17% of the markers were found to be unlinked. Loci of the configuration a/b x a/b and a/c x b/c were used to bridge seven homologous linkage groups between the two parents. The sex locus was not found to be associated with any linkage group. Key words : RFLP, bridge loci, repulsion phase linkage, double pseudotestcross.

Donor chromosome elimination and organelle composition of asymmetric somatic hybrid plants between an interspecific tomato hybrid and eggplant.

Morphology, the extent of elimination of donor chromosomes and the organelle composition of highly asymmetric somatic hybrid plants between a interspecific tomato hybrid Lycopersicon esculentum x L. pennellii (EP) as donor and a Solarium melongena, eggplant (E), recipient, were studied. Morphologically, the somatic hybrids most resemble eggplant but, due to polyploidy, growth is slower relative to both fusion parents. The somatic hybrids produce flowers that are characterized by abnormal styles, stigmas and by anthers which do not produce pollen. Limited amounts of donor EP genomic DNA were found in the three somatic hybrid plants (H18-1, H18-2 and H18-3), by dot-blot hybridization with probe pTHG2, equivalent to 6.23,5.41, and 5.95% EP, respectively. These percentages translated to the presence of 3.59, 2.90 and 3.19 average-size EP chromosomes in plants H1 8-1,-2 and-3, respectively. RFLP determination of L. esculentum- and L. pennellii-specific chromosomes revealed that only fragments of eight to ten out of the 24 EP chromosomes (EP has 12 L. esculentum and 12 L. pennellii chromosomes) are present in the asymmetric somatic hybrid plants. Loci of L. esculentum and L. pennellii were evenly represented in plants H18-1, -2, and -3: four to five from L. esculentum and four to five from L. pennellii. All somatic hybrid plants retained locus TG22, chromosome 4, from both EP species. Although the regeneration of plants, H18-1, -2 and-3 was from one callus, loci TG31 and TG79 of L. esculentum chromosome 2 and L. pennellii chromosome 9, respectively, were missing in hybrid plant H18-1. The three somatic hybrid plants all had chloroplast DNA fragments specific for S. melongena. The mitochondrial genome (mtDNA) in the asymmetric somatic hybrids showed predominantly the pattern of eggplant; however, some eggplant-specific polymorphic bands were not present in the three plants.

The role of irradiation dose and DNA content of somatic hybrid calli in producing asymmetric plants between an interspecific tomato hybrid and eggplant.

Highly asymmetric somatic hybrid plants were obtained by PEG/DMSO fusion of gamma-irradiated mesophyll protoplasts of the kanamycin-resistant (KmR(+)) interspecific hybrid Lycopersicon esculentum x L. pennellii (EP) with mesophyll protoplasts of Solanum melongena (eggplant, E). Elimination of the EP chromosomes was obtained by irradiating the donor genome with different doses of gamma rays (100, 250, 500, 750 and 1000 Gy). The selection of somatic hybrid calli was based on kanamycin resistance; EP and E protoplasts did not divide due to the irradiation treatment and sensitivity to kanamycin, respectively. KmR(+) calli were recovered following all irradiation doses of donor EP protoplasts. The hybrid nature of the recovered calli was confirmed by PCR amplification of the NptII gene, RAPD patterns and Southern hybridizations using potato ribosomal DNA and pTHG2 probes. Ploidy levels of calli confirmed as hybrid were further analyzed by flow cytometry. Such analyses revealed that the vast majority of hybrid calli that did not regenerate shoots were 5-9n polyploids. The three asymmetric somatic hybrid plants obtained were regenerated only from callus with a ploidy level close to 4n, and such calli occurred only when the donor EP had been exposed to 100 Gy. The amount of DNA in somatic hybrid calli, from 100-Gy exposure, was found by dot blot hybridization with the species-specific probe, pTHG2, to be equivalent with 3.1-25.8% of the tomato genome. Thus, DNA contained in 3.8-13.2 average-size tomato chromosomes was present in these hybrid calli. The asymmetric somatic hybrid plants had the eggplant morphology and were regenerated from one hybrid callus that contained an amount of tomato DNA equivalent to 6.29 average-size tomato chromosomes.

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

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.

T-DNA-tagged chromosome 12 in donor Lycopersicon esculentum × L. pennellii is retained in asymmetric somatic hybrids with recipient Solanum lycopersicoides.

Asymmetric somatic hybrid plants were recovered after fusing irradiated mesophyll protoplasts of donor Lycopersicon esculentum × L. pennellii (EP) interspecific hybrid with callus-derived protoplasts of recipient Solanum lycopersicoides. EP plant A54 had been previously transformed by an agrobacterium vector, and the T-DNA insert mapped to the L. esculentum chromosome 12. The T-DNA insert conferred kanamycin resistance to EP that was subsequently used to select cell fusion products and recover asymmetric hybrid plants that retained tagged chromosome 12. Doses of 50- and 100-Gy irradiation promoted the elimination of only a few donor chromosomes. At 200 Gy, the regenerated plants had ploidy levels higher than tetraploid. However, the T-DNA tagged chromosome 12 was always retained in the asymmetric hybrid plants tested. Likewise, all plants from the 100-Gy series, with the exception of number 160, were mixoploid in the root-tip cells. Such mixoploid asymmetric somatic hybrids could be stabilized by inducing adventitious shoots on leaf strips cultured on shoot regeneration medium containing kanamycin. The asymmetric hybrid plants did not produce viable seed when self-pollinated or backcrossed to tomato or S. lycopersicoides.

Cell type determines plastid transmission in tomato intergeneric somatic hybrids.

Mesophyll (M)- and suspension culture (S)-derived protoplasts of both Lycopersicon esculentum, tomato, and its wild relative Solanum lycopersicoides were fused as S + M, M + M and S + S combinations, respectively, to resolve the role of parental cell types in determining cpDNA transmission to intergeneric somatic hybrid plants. The mesophyll cpDNA was preferentially transmitted to 96% of the plants, each regenerated from a separate callus, in M + S and S + M fusion combinations. In contrast, for the M + M combination there was an equable distribution of either tomato cpDNA or that of S. lycopersicoides among the 34 hybrid plants. The number of plastids or proplastids in mesophyll or suspension protoplasts was not a factor regulating cpDNA transmission. Mesophyll or suspension protoplasts of both fusion partners had comparable frequencies of either plastid type with a mean of 23. The biased transmission of plastids from the mesophyll parent in somatic hybrid plants of S + M and M + S combinations appears to be due to differential multiplication of plastids, possibly conditioned by an unequal input of the nucleoids found in plastids versus proplastids. In the M + M fusion, plastid and nucleotide input and subsequent plastid multiplication are apparently equal, and when combined with random sorting out leads to an equal distribution of parental cpDNAs in the regenerated somatic hybrid plants. For the S+S combination, 22 somatic hybrid plants have exclusively tomato cpDNA, an outcome that is not readily explained by donor cell input.

Somatic embryogenesis in asparagus: the role of explants and growth regulators.

The explant used to initiate embryogenic callus and the growth regulators used in subsequent induction (IM) and embryo development media (EDM) both influenced rate of somatic embryo development and conversion to plantlets in asparagus. Embryogenic callus derived from spear-cross sections (SS), in vitro crowns (IVC) and lateral buds (LB) was cultured on IM of MS salts and vitamins with 2, 4-D or NAA at 0, 0.01, 0.1, 1.0 or 10 mg/l and kinetin at 0, 0.1, 1.0 or 10 mg/l. The auxin 2,4-D at 1-10 mg/l, in combination with kinetin at 0-1 mg/l, in IM induced the highest frequency of embryos after four weeks; callus derived from SS, IVC and LB had means of 394, 382, and 344 small globular embryos, and 4, 11 and 9 bipolar embryos per gram of callus, respectively. After 6 weeks on EDM, 128, 116 and 51 bipolar embryos (4-7 mm in length) occurred per gram callus and 4.5, 1.4 and 2.1 embryos converted for IVC, SS and LB, respectively. NAA at 1-10 mg/l, in combinations with kinetin 0-1 mg/l, yielded means of 64, 175 and 225 small globular embryos per gram callus on IM for SS, IVC and LB, respectively. NAA promoted a higher rate of embryo development: means of 27, 54 and 91 bipolar embryos per gram callus for SS, LB and IVC, respectively, on EDM. There were 0.5, 9.4 and 11.9 plantlets from these respective callus sources. There was no difference between kinetin levels of 0-1 mg/l on callus growth and embryogenesis, whereas, 10 mg/l in IM was inhibitory.

Somatic hybridization between selected Lycopersicon and Solanum species.

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 10(5)/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.

Interspecific somatic hybrid plants between eggplant (Solanum melongena) and Solanum torvum.

Mesophyll protoplasts of eggplant (cv Black Beauty) and of Solanum torvum (both 2n=2x=24) were fused using a modification of the Menczel and Wolfe PEG/DMSO procedure. Protoplasts post-fusion were plated at 1 × 10(5)/ml in modified KM medium, which inhibited division of S. torvum protoplasts. One week prior to shoot regeneration, ten individual calluses had a unique light-green background and were verified as cell hybrids by the presence of the dimer isozyme patterns for phosphoglucoisomerase (PGI) and glutamate oxaloacetate transaminase (GOT). Hybridity was also confirmed at the plant stage by DNA-DNA hybridization to a pea 45S ribosomal RNA gene probe. The ten somatic hybrid plants were established in the greenhouse and exhibited intermediate morphological characteristics such as leaf size and shape, flower size, shape, color and plant stature. Their chromosome number ranged from 46-48 (expected 2n=4x=48) and pollen viability was 5%-70%. In vitro shoots taken from the ten hybrid plants exhibited resistance to a verticillium wilt extract. Total DNA from the ten hybrids was restricted and hybridized with a 5.9 kb Oenothera chloroplast cytochrome f gene probe, a 2.4 kb EcoRI clone encoding mitochondrial cytochrome oxidase subunit II from maize and a 22.1 kb Sal I mitochondrial clone from Nicotiana sylvestris. Southern blot hybridization patterns showed that eight of ten somatic hybrids contained the eggplant cpDNA, while two plants contained the cpDNA hybridization patterns of both parents. The mtDNA analysis revealed the presence of novel bands, loss of some specific parental bands and mixture of specific bands from both parents in the restriction hybridization profiles of the hybrids.

Molecular analysis of single copy and repetitive genes on chromosome 2 in intergeneric tomato somatic hybrid plants.

Restriction fragment polymorphisms were used to identify and quantify the nuclear contributions from each parent to somatic hybrid plants between tomato (Lycopersicon esculentum Mill.) cv. Sub-Arctic Maxi and Solanum lycopersicoides Dun. Three single-copy clones, 2-13, 2-17, and 3-288, and a clone for the 45s ribosomal RNA, pHA2, all mapped to chromosome 2 of tomato, were used in analysis of 47 somatic hybrids. The amount of hybridizing probe for each parental band was quantified by densitometry of the autoradiograph film. Analyses with the three single-copy clones indicated that there were more than two S. lycopersicoides copies in most somatic hybrid plants. For at least one somatic hybrid there was a loss of one tomato copy. No evidence was found for more than two copies donated from tomato or loss of a copy from S. lycopersicoides. Most of the observed variation in copy number of the single-copy clones was consistent with chromosomal changes occurring in the suspension cells from which S. lycopersicoides parental protoplasts were derived.The number of copies of rDNA derived from each parent varied independently of the number of copies of single-copy clones from each parent. Changes in the copy number of rDNA occurred in both tomato and S. lycopersicoides genomes.

Plant regeneration from leaf protoplasts of Solanum torvum.

A protocol to obtain regenerated plants from protoplasts of Solanum torvum Sw a wild species of eggplant resistant to Verticillium wilt is reported. Leaf protoplasts were enzymatically isolated from six-week old seedlings grown in a controlled environment chamber. Protoplasts were plated on modified KM medium (0.4 M glucose)+(mg/l): 1.0 p-chlorophenoxyacetic acid (CPA)+1.0 naphthaleneacetic acid (NAA)+0.5 6-benzylaminopurine (BAP) and 0.02 abscisic acid (ABA). The protoplast density was 5×10(4) per ml with 5 ml placed in each of two quadrants in X-dishes (100×15 mm). The reservoir medium was modified KM+(mg/l): 0.1 NAA+0.5 BAP+0.1 M sucrose+0.1 M mannitol+0.6% washed agar+1% activated charcoal. Dishes were initially placed in the dark at 27°C. Protoplast division was initiated in 1-2 weeks and 4 weeks later p-calli were 1-3 mm. Plating efficiency was 11% when measured at 3 weeks. Six-week old p-calli were transferred individually onto Whatman No. 1 filter paper layered on modified KM (0.15 M sucrose)+mg/l: 2.0 indoleacetic acid (IAA)+2.0 zeatin+0.5% washed agar for 2 weeks. Subsequently, shoots occurred within 4 weeks at 70% efficiency on MS+30 g/l sucrose+2 mg/l zeatin. Shoots were rooted on half strength MS+10 g/l sucrose.

Somatic hybrid plants between Lycopersicon esculentum and Solanum lycopersicoides.

Leaf mesophyll protoplasts of Lycopersicon esculentum (2n=2x=24) were fused with suspension culture-derived protoplasts of Solanum lycopersicoides (2n=2x=24) and intergeneric somatic hybrid plants were regenerated following selective conditions. A two phase selection system was based on the inability of S. lycopersicoides protoplasts to divide in culture in modified medium 8E and the partial inhibition of L. esculentum protoplasts by the PEG/DMSO fusion solution. At the p-calli stage, putative hybrids were visually selected based on their hybrid vigor and lime-green coloration in contrast to slower growing parental calli characterized by a watery, whitish-brown coloration. Early identification of the eight hybrid plants studied was facilitated by isozyme analysis of leaf tissue samples taken from plants in vitro at the rooting stage. Regenerated plants growing in planting medium were further verified for hybridity by 5 isozymes marking 7 loci on 5 chromosomes in tomato. These included Skdh-1 mapped to chromosome 1 of tomato, Pgm-2 on chromosome 4, Got-2 and Got-3 on chromosome 7, Got-4 on chromosome 8, and Pgi-1 and Pgdh-2 both on chromosome 12. Fraction I protein small subunits further confirmed the hybrid nature of the plants with bands of both parents expressed in all hybrids. The parental chloroplasts could not be differentiated by the isoelectric points of the large subunit. Seven of the eight somatic hybrids had a chromosome number ranging from the expected 2n=4x=48 to 2n=68. Mixoploid root-tip cells containing 48, 53, 54 or 55 chromosomes for two of the hybrids were also observed.

A technique for bulk production of cytoplasts and miniprotoplasts from suspension culture-derived protoplasts.

Protoplasts isolated from suspension cultures of atrazine resistant black nightshade (Solanum nigrum L.) a weed biotype, were enucleated by centrifugation through a stepwise mannitol/sucrose gradient. Two cytoplast, enucleated subprotoplast, bands were routinely formed: one, a minor band at the 6.4%/18.2% mannitol border containing highly vacuolate cytoplasts with 95%+ enucleation; secondly a major cytoplast band at the 18.2% mannitol/33% sucrose border containing 90%+ enucleated protoplasts in quantities up to 4 million per 50 ml gradient tube. Efficient production of cytoplasts depended on the subculture procedures used for the cell suspensions. Optimal cytoplast yield (44%) occurred for protoplasts isolated three days after subculture. The vigor of the donor suspension cultures as visually monitored had to be controlled in order to obtain consistently high enucleation percentages.

Differential fate of plastid and mitochondrial genomes in Petunia somatic hybrids.

The chloroplast (cp) and mitochondrial (mt) DNAs of Petunia somatic hybrid plants, which were derived from the fusion of wild-type P. parodii protoplasts with albino P. inflata protoplasts, were analyzed by endonuclease restriction and Southern blot hybridization. Using (32)P-labelled probes that distinguished the two parental cpDNAs at a BamH1 site and at a HpaII site, only the P. parodii chloroplast genome was detected in the 10 somatic hybrid plants analyzed. To examine whether cytoplasmic mixing had resulted in rearrangement of the mitochondrial genome in the somatic hybrids, restriction patterns of purified somatic hybrid and parental mtDNAs were analyzed. Approximately 87% of those restriction fragments which distinguish the two parental genomes are P. inflata-specific. Restriction patterns of the somatic hybrid mtDNAs differ both from the parental patterns and from each other, suggesting that an interaction occurred between the parental mitochondrial genomes in the somatic fusion products which resulted in generation of the novel mtDNA patterns. Southern blot hybridization substantiates this conclusion. In addition, somatic hybrid lines derived from the same fusion product were observed to differ in mtDNA restriction pattern, reflecting a differential sorting-out of mitochondrial genomes at the time the plants were regenerated.

Expression of nuclear-cytoplasmic genomic incompatibility in interspecific Petunia somatic hybrid plants.

Somatic hybrid plants were regenerated following calcium-high pH fusion of the unidirectional, sexually incompatible cross of Petunia parodii wild-type leaf mesophyll protoplasts with protoplasts from a cytoplasmic determined chlorophyll-deficient mutant of P. inflata. Genic complementation to chlorophyll synthesis and sustained growth in the selective medium was used to visually identify hybrid calluses. Hybrid calluses were subsequently regenerated to shoots, rooted, and confirmed as somatic hybrids by their intermediate floral and leaf morphology based on comparison to the 2 n = 4 x = 28 sexual counterpart, dominant anthocyanin expression in the corolla, chromosome number, and peroxidase and maleic dehydrogenase isozyme patterns. Certain cytologically stable somatic hybrids displayed aberrant reproductive and floral morphologies including subtle to moderate corolla and leaf pigment variegation, floral dimension changes and reduced pollen viability. In contrast, cytologically unstable somatic hybrids showed various degrees of aneuploidy coupled with corolla splitting, and irregularities in reproductive organs such as double stigmas and styles in addition to reduced pollen viability. Postulated mechanisms to account for these phenotypic changes in stable and unstable somatic hybrids include nuclear-cytoplasmic genomic incompatibility, chromosome loss in a biparental cytoplasm, or a phenomenon similar to hybrid dysgenesis occurring as a result of somatic fusion.

Medium components for shoot cultures of chlorophyll-deficient mutants of petunia inflata.

Selected medium components were tested for 30 day growth promotion of shoot tips of Petunia inflata wild type, a cytoplasmic and a nuclear inherited chlorophyll-deficient mutant. Experiments were conducted independently with iron, sucrose, thiamine-HCl, indole-3-acetic acid (IAA), Kinetin (K), 6-benzylaminopurine (BA), coconut milk (CM), casein hydrolysate (CH), and plant extract (PE) an aqueous leaf extract, added to modified Murashige and Skoog (MS) salts and vitamins medium, and pH between 4.0 and 7.0 was also compared. The optimum concentrations of all test components were used to formulate revised MS media especially designed for in vitro shoot growth of chlorophyll-deficient petunia mutants. The optimum medium for the nuclear albino was: MS salts+1.5 mg/l thiamine-HCl+100 mg/l myo-inositol+3.5% sucrose+1% PE+5.0 mg/l IAA+0.3 mg/l K at pH 6.0; for the wild type: MS salts+0.6 mg/l thiamine-HCl+100 mg/l myo-inositol+4% sucrose+1.0 mg/l IAA+0.3 mg/l K at pH 5.0 and for the cytoplasmic albino: MS salts+0.4 mg/l thiamine-HCl+100 mg/l myo-inositol+4% sucrose+20% CM+3% PE+1.0 mg/l K at pH 5.0. On the revised MS media a 3-, 4- and 5- fold increase in 30 day plant fresh weight occurred for the nuclear, wild type and cytoplasmic chlorophyll-deficient plants, respectively.