Somatic hybridization of sexually incompatible petunias: Petunia parodii,Petunia parviflora

Summary Somatic hybrid plants were regenerated following the fusion of leaf mesophyll protoplasts of P. parodii with those isolated from a nuclear-albino mutant of P. parviflora. Attempts at sexual hybridization of these two species repeatedly failed thus confirming their previously established cross-incompatibility. Selection of somatic hybrid plants was possible since protoplasts of P. parodii would not develop beyond the cell colony stage, whilst those of the somatic hybrid and albino P. parviflora produced calluses. Green somatic hybrid calluses were visible against a background of albino cells/calluses, and upon transfer to regeneration media gave rise to shoots. Shoots and the resultant flowering plants were confirmed as somatic hybrids based on their growth habit, floral pigmentation and morphology, leaf hair structure, chromosome number and Fraction 1 protein profiles. The relevance of such hybrid material for the development of new, and extensively modified cultivars, is discussed.     

Differential fate of plastid and mitochondrial genomes in Petunia somatic hybrids

Summary 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 ³²P-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.     

Fraction I protein analysis of parasexual hybrid plants Arabidopsis thaliana + Brassica campestris

The polypeptide composition of Fraction I protein (ribulose-1,5-bisphosphate carboxylase) prepared from leaves of two clones of the parasexual hybrid plant Arabidopsis thaliana + Brassica campestris as well as their parents was analyzed by isoelectric focusing. The protein in hybrid plants contained a heterogenous population of small subunits resulting from the expression of both Arbabidopsis and Brassica nuclear genes, whereas the large subunit polypeptides, and hence the functional chloroplast DNA, were from the Brassica parent.     

Somatic hybrids between unilateral cross-incompatible Petunia species

Summary Somatic hybrid plants regenerated following the fusion of leaf mesophyll protoplasts of Petunia parodii with those isolated from a cell suspension of albino P. inflata. These two species exhibit a unilateral cross-incompatability with a pre-zygotic mode of reproductive isolation preventing hybridizations with P. inflata as the maternal parent. Selection of somatic hybrids relied on the fact that unfused or homokaryon protoplasts of P. parodii did not develop beyond the cell colony stage while those of the putative somatic hybrids and albino P. inflata parent produced callus. Green somatic hybrid calluses were readily identified against the white background of P. inflata following complementation to chlorophyll synthesis proficiency and continued growth in hybrid cells. Shoots, and ultimately flowering plants, were identified as somatic hybrids based on their floral morphology and colour, chromosome number and the fact that they segregated for parental characters. The frequency of somatic hybrid production was comparable to that previously established for two sexually compatible Petunia species.     

Expression of nuclear and chloroplastic genes coding for fraction-1 protein in somatic hybrids of Nicotiana tabacum + rustica

In the sexual interspecific cross, Nicotiana rustica L.xN. tabacum L., N. rustica can serve as the female but not as the male parent. By fusion of protoplasts, the barrier to fertilization was overcome and somatic hybrids containing N. tabacum cytoplasm were produced as shown by isoelectric focusing of the Fraction-1 protein (F-1-protein). All somatic hybrids displayed polypeptides of the large subunit of F-1 protein (which is coded by the chloroplast genome) characteristic of only one or the other parental species. Two hybrids had large subunits of the N. tabacum type and two hybrids had those of the N. rustica type. Three hybrids contained three smallsubunit polypeptides (coded by the nuclear genome), one being characteristic of N. rustica, one characteristic of N. tabacum, and one with an isoelectric point common to both species. A fourth hybrid contained only two small-subunit polypeptides of the N. tabacum type but in a F-1 protein macromolecule whose large subunits were of the N. rustica type. One somatic hybrid was self-fertile and its F₂ progeny contained large- and small-subunit polypeptides indistinguishable in their isoelectric points from those in the parent F₁ hybrid. All somatic hybrids showed an aneuploid chromosome number and morphological characteristics intermediate between those of N. rustica and N. tabacum.     

Restriction endonuclease analysis of chloroplast DNA in interspecies somatic Hybrids of Petunia

Summary Restriction endonuclease cleavage pattern analysis of chloroplast DNA (cpDNA) of three different interspecific somatic hybrid plants revealed that the cytoplasms of the hybrids contained only cpDNA of P. parodii. The somatic hybrid plants analysed were those between P. parodii (wild type) + P. hybrida (wild type); P. parodii (wild type)+P. inflata (cytoplasmic albino mutant); P. parodii (wild type) + P. parviflora (nuclear albino mutant). The presence of only P. parodii chloroplasts in the somatic hybrid of P. parodii + P. inflata is possibly due to the stringent selection used for somatic hybrid production. However, in the case of the two other somatic hybrids P. parodii + P. hybrida and P. parodii + P. parviflora it was not possible to determine whether the presence of only P. parodii chloroplasts in these somatic hybrid plants was due to the nature of the selection schemes used or simply occurred by chance. The relevance of such somatic hybrid material for the study of genomic-cytoplasmic interaction is discussed, as well as the use of restriction endonuclease fragment patterns for the analysis of taxonomic and evolutionary inter-relationships in the genus Petunia.     

Morphological and molecular characterization of fertile tetraploid somatic hybrids produced by protoplast electrofusion and PEG-induced fusion between Lycopersicon esculentum Mill. and Lycopersicon peruvianum Mill.

Summary Mesophyl protoplasts of two genotypes of cultivated tomato (Lycopersicon esculentum Mill.) and one of its wild relative species (Lycopersicon peruvianum Mill.) were fused by using electrofusion and polyethyleneglycol-induced fusion. Forty-three fertile tetraploid somatic hybrid plants, each deriving from separate calli, were recovered from both fusion procedures. Electrofusion appeared more efficient than chemical fusion for the production of somatic hybrids. These plants appeared morphologically similar, whatever the fusion procedure and tomato genotype. They had intermediate leaf, inflorescence, and flower morphology. After self-pollination, the hybrids set fruit of intermediate size and color. The hybrid nature of these plants was confirmed by isoelectric focusing of the Rubisco small subunits used as nuclear markers. L. esculentum and L. peruvianum were distinguished by means of two chloroplast markers: CF1-ATPase subunit as analyzed by isoelectro-focusing and ct DNA restriction patterns. All hybrids displayed both ct markers of only one parent with no biased transmission. Mitochondrial (mt) DNAs were prepared from flower buds by using miniaturized CsCl gradients. Preliminary analysis indicated that mt genomes from the hybrids all differed from those of both parents. mt DNA Sall restriction enzyme analysis revealed that all but two hybrids contained one novel fragment of 13.5 kb. Gene mapping experiments showed that the mt apocytochrome b and ATPase subunit 9 homologies in the somatic hybrid mt DNA resembled L. esculentum and L. peruvianum, respectively; the mt nad5 probe distinguished at least four distinct patterns in the hybrids. These results indicated that mt DNA rearrangements involving intergenomic recombinations occurred through protoplast fusion. A greater mt DNA polymorphism was induced with chemical fusion than with electrofusion.     

The evolution of fraction I protein during the origin of a new species ofNicotiana

The polypeptide composition of Fraction I protein from Nicotiana digluta, a synthetic species which arose by chromosome doubling following the interspecific hybridization of N. glutinosa and N. tabacum, has been examined by isoelectric focusing. The composition of the protein from N. digluta, which was identical to the protein from the infertile F1 hybrid N. glutinosa x N. tabacum, showed 3 polypeptides in the large subunit and 4 polypeptides in the small subunit. The large subunit polypeptides were identical to those from N. glutinosa, the maternal parent in the original hybridization, whereas the small subunit polypeptides were a composite of the small subunit polypeptides from both N. glutinosa and N. tabacum. This analysis demonstrates how the polypeptide composition of Fraction I protein evolves during the origin of new species of Nicotiana.     

Evolution of fraction 1 protein in the genus Lycopersicon

The large- and small-subunit polypeptide composition of fraction 1 protein contained in seven species of Lycopersicon and Solanum pennellii was determined by electrofocusing. The eight species of protein had large subunits composed of three polypeptides separated by about 0.05 pH unit, but there was no difference in the isoelectric points of the clusters of three polypeptides. By this criterion, no surviving mutations have appeared in the extranuclear DNA coding for the cluster of large-subunit polypeptides during a period of evolution which generated the eight species of plants. The genus Lycopersicon appears to be much younger than its sister genus Nicotiana in the family Solanaceae, where four types of polypeptide clusters have evolved. Three different small-subunit polypeptides whose isoelectric points are coded by nuclear DNA have arisen among the seven Lycopersicon species, and L. hirsutum and S. pennellii have proteins containing single polypeptides and are therefore considered older than L. chilense, L. chimielewskii, and L. parviflorum, whose proteins contain two polypeptides. L. cheesemanii, L. pimpinellifolium, and L. esculentum (and probably L. peruvianum) seem to be the most recently evolved species since their fraction 1 proteins have small subunits composed of three polypeptides.This research was supported by NSF Grant 75-07368 and Contract No. EY-76-S-03-0034, P. A. #8, from the Department of Energy.     

Somatic hybridization between male sterile Nicotiana tabacum and N. glutinosa through protoplast fusion

Summary Protoplasts derived from suspension cultured cells of cytoplasmic male sterile Nicotiana tabacum (N. debneyi cytoplasm) and of fertile N. glutinosa were fused with the aid of polyethylene glycol (PEG). Out of 1,089 colonies developed from PEG-treated protoplasts, 29 restored whole plants.A somatic hybrid plant was selected on the basis of isoelectrofocusing analysis of Fraction I protein in leaves of regenerated plants. A newly created hybrid contained small subunits of both parents but only a N. glutinosa type large subunit.Male sterile character was conserved in a hybrid plant while leaf morphology was intermediate between the parents. By tobacco mosaic virus infection tests, the hybrid's leaves showed resistant symptoms, hypersensitive local lesions, which were due to N. glutinosa nuclear genome expression.Abbreviations PEG Polyethylene glycol - TMV Tobacco mosaic virus     

Detection of the Nicotiana rustica chloroplast genome coding for the large subunit of Fraction I protein in a somatic hybrid in which only the N. tabacum chloroplast genome appeared to have been expressed

Nine plants were produced from anthers of a somatic hybrid which had been obtained by fusion of Nicotiana tabacum L. and N. rustica L. protoplants. As determined by electrofocusing, the Fraction I protein of the original somatic hybrid had largesubunit polypeptides exclusively of the N. tabacum type. Two of the plants regenerated from anthers contained Fraction-I-protein large subunits exclusively of the N. rustica type. Since each plant was regenerated from a single cell, the somatic hybrid must have had cells containing both the N. tabacum and N. rustica chloroplast genome although the latter was not expressed. Possibilities to account for this non-expression of a chloroplast genome in the somatic hybrid are discussed.     

Fertile somatic hybrids between Petunia hybrida and a wild species,Petunia variabilis

Somatic hybrid plants were regenerated following electrofusion between leaf mesophyll protoplasts of P. hybrida (2n = 14) and a wild sexually incompatible species, P. variabilis (2n = 18). The selection of hybrids was based on the hybrid vigour, expressed both in the growth of the callus and at the shoot formation stage, resulting from the combination of parental genomes. Calli exhibiting vigorous growth were selected, and upon transfer to regeneration medium gave rise to shoots. Four regenerated plants from three calli had morphological characteristics intermediate between those of the parents. The hybrid nature of these plants was confirmed by chromosome counts as well as isozyme and DNA analyses. They had amphidiploid chromosome numbers (2n = 32) and were fertile. Following self-pollination and backcrossing with P. variabilis, large numbers of F₂ and BC₁ seedlings were obtained.     

Somatic hybrid plants between Lycopersicon esculentum and Solanum lycopersicoides

Summary 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.Michigan Agricultural Experiment Station Journal Article No. 11736. Supported by Grant No. I-751-84R from BARD — The United States — Israel Binational Agricultural Research and Development Fund     

Nontranslation of foreign genetic information for fraction 1 protein under circumstances favorable for direct transfer ofNicotiana gossei isolated chloroplasts intoN. Tabacum protoplasts

Summary The nuclei and cytoplasm ofN. gossei andN. tabacum are compatible to the extent that reciprocal, interspecific F₁ hybrids can be produced by conventional breeding techniques. Conditions were established in which manyN. gossei isolated chloroplasts could be seen by phase and fluorescence microscopy to adhere to 40% of the population of protoplasts obtained from white tissue of variegatedN. tabacum plants and to remain attached after washing the protoplasts. Chloroplasts also could be seen to enter the interior of the protoplasts. After treating albino protoplasts withN. gossei chloroplasts, the protoplasts were subjected to further conditions whereby 65 calluses containing shoots developed. TwentyN. tabacum protoplasts not treated with foreign chloroplasts also produced calluses with shoots to serve as a control. All calluses developed chlorophyll irrespective of whether or not the albino protoplasts had been treated with isolatedN. gossei chloroplasts. The Fraction 1 protein ofN. tabacum has a different electrophoretic mobility from the protein ofN. gossei or anN. gossei xN. tabacum F₁ hybrid. The Fraction 1 protein large subunit is coded by chloroplast DNA, whereas the small subunit is coded by nuclear DNA. Fraction 1 protein was isolated from the variegated shoots of the 65 calluses obtained after treating albino protoplasts with foreign chloroplasts. Immunoelectrophoresis demonstrated the protein from each callus to have a mobility identical toN. tabacum protein. Therefore, under circumstances highly favorable for the direct transfer ofN. gossei isolated chloroplasts (and possibly nuclei also) intoN. tabacum protoplasts, no evidence was obtained to suggest that genetic information contained in the isolated foreign organelles was being translated into the polypeptides of either the large or small subunits of Fraction 1 protein contained in newly differentiated leaves derived from the protoplasts. Supported by Research Grant PCM-75-07368 from the National Science Foundation.     

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

Summary 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.Michigan Agricultural Experiment Station Journal Article No. 11376. Supported by Grant No. I-134-79 from BARD — The United States — Israel Binational Agricultural Research and Development Fund, and by grant 11-77-4 from American Florists Endowment     

A comparison of the polypeptide isoelectric points and antigenic determinant sites of the large subunit of fraction 1 protein from Lycopersicon esculentum, Nicotiana tabacum and Petunia hybrida.

The large subunit of Fraction 1 protein from Lycopersicon esculentum, Nicotiana tabacum and Petunia hybrida has been examined by isoelectric focusing of the S-carboxymethylated polypeptides, and by double immunodiffusion with antiserum raised against Fraction 1 protein. The immunological results reveal heterogeneity in the large subunit primary structure not identified by isoelectric focusing. A variable phylogeny can be generated depending on whether serological or electrofocusing criteria are used.     

Differentiation of Fraction 1 protein in relation to age and distribution of Angiosperm groups

Fraction 1 protein (F-1-protein) (ribulose bisphosphate carboxy-lase-oxygenase) contained inLemnaceae has been evolving for at least 50 million years because fossils of these plants have been identified in strata belonging to the Upper Cretaceous. Electrofocusing F-1-protein resolves the large subunit polypeptides coded by extranuclear DNA and the small subunit polypeptides coded by nuclear DNA. Four differences affecting isoelectric points of the large subunit polypeptides and eight affecting the small subunit polypeptides are now present among eleven species representing the four genera comprising theLemnaceae. In comparison, four differences in the large and 13 in the small subunit polypeptides exist among 63 species ofNicotiana; four differences in the large and eight differences in the small subunit polypeptides exist among 19 species ofGossypium. The number of differences in F-1-protein composition being of the same order of magnitude for the generaNicotiana, Gossypium, and the familyLemnaceae, we infer that these Angiosperms are of similar antiquity. Nicotiana species indigenous to Australia and Africa contain F-1-proteins whose large subunit polypeptides are different but some of whose small subunit polypeptides are like those found in species from the Western Hemisphere. The same situation is found for the F-1-protein inGossypium. These results are in harmony with the view that species ofNicotiana andGossypium have arrived in Australia via former land connections between S. America, Antarctica, and Australia.     

The Partial Characterization of the Major Seed Storage Proteins in Arabidopsis thaliana L.

This study was aimed at the characterization of the major storage proteins in Arabidopsis thaliana. Two major protein fractions, i.e., the fraction Ⅰ and Ⅱ proteins, were isolated from the extract of mature seeds of this plant by molecular seive gel filtration chromatography. Various polyacrylarnide gel electrophoretic techniques were used to study the properties and polypeptide compositions of these two protein fractions. In was shown that during the SDS gel electrophoresis, fraction Ⅰ protein was separated into 6 major bands with the mol. was. of 34, 31, 29, 28 and 19-20 kD, respectively, whereas Fraction Ⅱ protein migrated as 3 low mol. wt. bands (10-12 kD) on the same gel. Non-denaturing native gel electrophoresis revealed that fraction Ⅰ was a neutral protein and Fraction Ⅱ was a positively charged basic protein with an isoelectric point (pI) higher than 8.8. Fraction I protein was further separated into at least 16 polypeptides in isoelectric focusing/SDS two-dimensional gel electrophoresis, i.e. each SDS band contained 3-4 polypeptides with the same mol. wt. but different pis. This suggested a more complex polypeptide composition of this protein. The properties of fraction Ⅰ and Ⅱ proteins were in good accordance with that of the 12s and 1.7s storage globulins in seeds of many other dicotyledonous plants, and therefore had been characterized as the two major seed storage proteins in this species. These two storage globulins were shown to be accumulated within a defined period during the late stage of seed development (12-14 DAF) and became predominant protein components in mature seeds. In the mean time, a few points in relation to the polypeptide composition and subunit molecular configuration of the 12s globulin were noted.     

A simple electrophoretic procedure for the determination of the polypeptide composition of the subunits of Fraction 1 protein

A rapid and convenient method is described for resolving the polypeptide composition of Fraction 1 protein. Using crude leaf extracts of a number of Lycopersicon species, Fraction 1 protein was first separated by polyacrylamide gel electrophoresis and the gel slices containing the protein were isoelectrofocused in the presence of 8 m urea. Isoelectric focusing was also applied directly on subunits in gel slices obtained after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The polypeptide composition produced is in agreement with previous determinations obtained by more elaborated techniques.     

The interspecific hybrid Petunia parodii × P. inflata and its relevance to somatic hybridization in the genus Petunia

Summary Attempts at the reciprocal cross between Petunia parodii and P. inflata using standard emasculation and pollination techniques failed. Limited pollen tube growth down the style in reciprocal crosses led to reproductive isolation between the self-compatible P. parodii and self-incompatible P. inflata. The interspecific hybrid was successfully produced by bud-pollination of P. parodii with P. inflata as the male parent in 22 percent of attempts, but not in the opposite direction. In vitro pollination of P. parodii ovaries with P. inflata pollen also produced hybrids. The small size of the ovary made it technically impossible to use P. inflata as the female parent for in vitro pollination. The interspecific hybrids were intermediate, as compared to the two parents, for six of the seven plant and flower characters measured. Furthermore, the hybrids had high pollen fertility, set abundant seed upon self-pollination, and readily inter-crossed with the parental species. The results are consistent with a high degree of chromosomal homology in the parental species and with minor genetic divergency leading to reproductive isolation that is pre-zygotic in nature. Overcoming the barriers to cross-incompatibility by practical techniques resulted in fertile interspecific hybrids that segregated for parental characters. The potential value of employing the parental species in somatic hybridization experiments is discussed.Michigan Agricultural Experiment Station Journal Article No. 8404