Dvora Aviv

The I2C family from the wilt disease resistance locus I2 belongs to the nucleotide binding, leucine-rich repeat superfamily of plant resistance genes.

Characterization of plant resistance genes is an important step in understanding plant defense mechanisms. Fusarium oxysporum f sp lycopersici is the causal agent of a vascular wilt disease in tomato. Genes conferring resistance to plant vascular diseases have yet to be described molecularly. Members of a new multigene family, complex I2C, were isolated by map-based cloning from the I2 F. o. lycopersici race 2 resistance locus. The genes show structural similarity to the group of recently isolated resistance genes that contain a nucleotide binding motif and leucine-rich repeats. Importantly, the presence of I2C antisense transgenes abrogated race 2 but not race 1 resistance in otherwise normal plants. Expression of the complete sense I2C-1 transgene conferred significant but partial resistance to F. o. lycopersici race 2. All members of the I2C gene family have been mapped genetically and are dispersed on three different chromosomes. Some of the I2C members cosegregate with other tomato resistance loci. Comparison within the leucine-rich repeat region of I2C gene family members shows that they differ from each other mainly by insertions or deletions.

Interspecific Transfer of Cytoplasmic Male Sterility by Fusion between Protoplasts of Normal Nicotiana sylvestris and X-Ray Irradiated Protoplasts of Male-Sterile N. tabacum

Summary X-irradiated protoplasts from plants of a cytoplasmic male-sterile (CMS) cultivar of Nicotiana tabacum ( N. suaveolens cytoplasm) were fused with protoplasts of N. sylvestris plants. The selection of fusion products was based on the suppression of cell division by X-irradiation of the CMS ≪parent≫ protoplasts and on the use of mannitol medium, which is unfavorable to protoplasts of N. sylvestris . The protoplast ≪feeder-layer≫ technique was utilized in order to rescue the low frequency of fusion products which withstood the selection procedure. Thirty-one mature plants, regenerated from 7 calli, were obtained. Of these, 21 plants (Type A) had the shoot, leaf and perianth morphologies of N. sylvestris but their anthers resembled the anthers of the CMS N. tabacum ≪parent≫ and in fact all were male-sterile. Of these, 7 plants had 2n = 24 chromosomes and 14 plants had 2n = 48 chromosomes. Six plants (Type B) grew as rosettes and bore flowers with white corollas, i. e., similar to N. sylvestris , but had deformed leaves and during further growth were similar to N. tabacum; their anthers were sterile but normal, or almost normal in structure of these, 4 plants had 2n = 48 chromosomes and 2 plants had 2n = 76 to 80 chromosomes. Five plants (Type C), all resulting from one callus, were identical in shoot, leaf and floral morphology to the cytoplasmic male-sterile N. tabacum ≪parent≫ These plants are considered to be the results of a cytoplasmic male-sterile N. tabacum protoplast which ≪escaped≫ the X-irradiation effect. These results led to the conclusion that Type A plants bore the genome of N. sylvestris while their plastome was partially or entirely of the CMS ≪parent≫ (i.e., of N. suaveolens ). These Type A plants, which are the majority among the fusion products, are therefore considered somatic hybrids resulting from the transfer of male-sterility-causing cytoplasm into N. sylvestris cells. The genetic composition of Type B plants is uncertain. They probably have a mostly N. sylvestris genome but also some N. tabacum genes; their cytoplasm is probably not pure N. sylvestris . This protoplast fusion procedure may have a general applicability for interspecific transfer of cytoplasmic male-sterility, as well as other cytoplasmic factors which are hard, if not impossible, to transfer by classical genetic techniques.

Enhanced oxidative-stress defense in transgenic potato expressing tomato Cu,Zn superoxide dismutases.

SummaryThe two cDNAs coding for the cytosolic (cyt) and the chloroplast-located (chl) Cu,Zn superoxide dismutases (SODs) of tomato (Perl-Treves et al. 1988) were cloned into respective binary vectors and mobilized into Agrobacterium strains. Potato tuber discs were infected with either of the two agrobacterial strains and cultured on selective medium containing kanaymcin. The integration of either of the cyt or the chl SOD transgenes was verified by Southern-blot hybridization. The enzymatic activity of the additional tomato chl Cu,Zn SOD could be distinguished from endogenous SOD activity since the latter isozyme migrated faster on SOD-activity gels. Several transgenic potato lines harboring either the cyt or the chl SOD genes of tomato showed elevated tolerance to the superoxide-generating herbicide paraquat (methyl viologen). After exposure of shoots to paraquat, tolerance was recorded either by scoring symptoms visually or by measurements of photosynthesis using the photoacoustic method. Root cultures from transgenic lines that harbored the additional cyt Cu,Zn SOD gene of tomato were tolerant to methyl viologen up to 10-5 M; a lower tolerance was recorded in roots of transgenic lines that expressed the additional chl Cu,Zn SOD of tomato.

Cytoplasmic hybridization in Nicotiana: Mitochondrial DNA analysis in progenies resulting from fusion between protoplasts having different organelle constitutions

SummaryOur previous studies indicated that fusion products with one functional nucleus but organelles of the two fusion partners (i.e. heteroplastomic cybrids) could be obtained by fusing X-irradiated (cytoplasmic donor) with non-irradiated (recipient) Nicotiana protoplasts. The present report deals with the analysis of mitochondria in cybrid populations resulting from the fusion of donor Nicotiana tabacum protoplasts with recipient protoplasts having a N. Sylvestris nucleus but chloroplasts of an alien Nicotiana species, and exhibiting cytoplasmic male sterility. The two fusion parents showed significant differences in restriction patterns of their chloroplast and mitochondrial DNA. Four groups of cybrid plants were obtained by this fusion. All had N. sylvestris nuclei but contained either donor or recipient chloroplasts and had either sterile or fertile anthers. There was no correlation between anther fertility and chloroplasts type. The mitochondrial DNA restriction patterns of sterile cybrids were similar to the respective patterns of the sterile fusion partner while the mitochondrial DNA restriction patterns of the fertile cybrids were similar to the respective patterns of the fertile fusion partner. The results indicate an independent assortment of chloroplasts and mitochondria from the heteroplastomic fusion products.

Progeny analysis of the interspecific somatic hybrids; Nicotiana tabacum (CMS) + Nicotiana sylvestris with respect to nuclear and chloroplast markers.

SummaryThe progeny of a fusion experiment involving N. sylvestris protoplasts and X-irradiated protoplasts of the cytoplasmic male sterile ‘Line 92’ (N. tabacum nucleus and alien, male-sterility inducing, cytoplasm) were analyzed. Three groups of somatic hybrid plants resulted: Type A, Type B-1 and Type B-2. These as well as their androgenic progenies and the progenies resulting from their pollination with N. tabacum or N. sylvestris were followed with respect to several nuclear and cytoplasmic traits. Those controlled by the nuclear genome were plant and flower morphologies; those controlled by genetic information in the cytoplasm were tentoxin sensitivity (affecting the coupling factor of chloroplast ATPase), the large subunit of ribulose bisphosphate carboxylase and the restriction endonuclease pattern of plastid DNA. A further cytoplasmic trait investigated (exact site of genetic control not known) was male sterility. The examinations of the somatic-hybrid groups and their respective progenies indicated that: Type A plants have N. sylvestris nuclei and ‘Line 92’ plastids; Type B-1 plants also have ‘Line 92’ plastids but their genome is composed of N. sylvestris and N. tabacum nuclei; Type B-2 plants with impaired male fertility had N. sylvestris plastids and N. sylvestris nuclei.

Restoration of fertility in cytoplasmic male sterile (CMS) Nicotiana Sylvestris by fusion with X-irradiated N. tabacum protoplasts.

SummaryRestoration of male fertility was achieved by fusing protoplasts from male sterile (CMS) Nicotiana sylvestris plants with X-irradiated protoplasts derived from fertile N. tabacum plants. The CMS N. sylvestris plants were derived from a previous somatic hybridization experiment and contained alien (Line 92) cytoplasm. About one quarter of the regenerated plants were found to be cybrids. i.e. they consisted of N. sylvestris nuclei combined with all or some components of N. tabacum cytoplasm. In one half of these cybrids male fertility was restored to different levels. The chloroplasts of the two parental donors differ in respect to tentoxin sensitivity: chloroplasts of CMS N. sylvestris are sensitive while those of N. tabacum are insensitive. It could therefore be demonstrated that there was an independent segregation of chloroplast type and male fertility/sterility: several somatic cybrids were male fertile but tentoxin sensitive and others were tentoxin insensitive yet they were male sterile. Only in about one half of the somatic cybrids was male fertility restored together with restoration to tentoxin insensitivity.

Genetic transformation of Citrus protoplasts and regeneration of transgenic plants.

Abstract Citrus protoplast-derived embryogenic calli can readily be induced to differentiate embryos which further develop into mature fruit bearing trees. We successfully introduced plasmid pCAP212 DNA, harboring the coding sequences of neomycin phosphotransferase (nptII) and chloramphenicol acetyltransferase (cat) genes into Citrus protoplasts via polyethylene glycol (PEG) treatment resulting in transgenic plants. Transient expression of chloramphenicol acetyltransferase (CAT) activity was detected 3 days after the direct transformation was performed. Microcolonies (0.5 mm in diameter) were selected on agar medium containing paromomycin sulfate (PAR). Twenty one resistant clones, presumably containing the nptII gene, were isolated. To verify the transgenic nature of the isolated clones either neomycin phosphotransferase (nptII) activity was measured or Southern hybridization was performed. Altogether 9 positive stably transformed embryogenic clones were obtained; two of these were regenerated into transgenic rooted plants.

Isolation of two cDNA clones from tomato containing two different superoxide dismutase sequences

A cDNA library was derived from the poly(A)+ RNA of young tomato leaves. The library was cloned in a λgt11 system and screened by synthetic oligonucleotide probes having sequences that match the codes of conserved regions of amino acid sequences of Cu,Zn superoxide dismutase (SOD) proteins from a wide range of eukaryotic organisms. Two cDNAs were isolated, cloned and sequenced. One of the cDNAs, P31, had a full-size open reading frame of 456 bp with a deduced amino acid sequence having an 80% homology with the deduced amino acid sequence of the cytosolic SOD-2 cDNA of maize. The other cDNA, T10 (extended by T1), had a 651 bp open reading frame that revealed, upon computer translation, 90% homology to the amino acid sequence of mature spinach chloroplast SOD. The 5′ end of the reading frame seems to code for a putative transit peptide. This work thus suggests for the first time an amino acid sequence for the transit peptide of chloroplast SOD. Northern hybridizations indicated that each of the P31 and T10 clones hybridized to a blotted poly(A)+ RNA species. These two species are differentially expressed in the plant organs: e.g., the species having the T10 sequence was detected in the leaves but not in roots, while the one with the P31 sequence was expressed in both leaves and roots. The cDNA clones P31 and T10 were also hybridized to Southern blots of endonuclease fragmented tomato DNA. The clones hybridized to specific fragments and no cross hybridization between the two clones was revealed under stringent hybridization conditions; the hybridization pattern indicated that, most probably, only one locus is coding for each of the two mRNA species.

Cybrids containing mixed and sorted-out chloroplasts following interspecific somatic fusions in Nicotiana

SummaryStreptomycin resistance was transferred by “donor-recipient” protoplast fusion from Nicotiana tabacum (SR-1) protoplasts into Nicotiana tabacum (cytoplasmic male sterile — Line 92) protoplasts in one case and into Nicotiana sylvestris protoplasts in another. It is demonstrated that streptomycin resistance (SR-1) is a chloroplast marker which segregates independently from a mitochondrial marker.In the fusion experiment where Nicotiana tabacum (Line 92) was the recipient, microcalli were plated in the presence of streptomycin. In this case, chloroplast sorting out occurred at a stage preceeding plant regeneration, producing stable streptomycin resistant cybrids. In the fusion whre Nicotiana sylvestris was the recipient, no direct selection for streptomycin resistance was performed. In this case chloroplast sorting out was incomplete, thus producing cybrid plants with a mixed chloroplast population. In some plants, sorting out of streptomycin resistant and sensitive chloroplasts was still apparent in the second generation progeny.

Ethylene and in vitro culture of potato: suppression of ethylene generation vastly improves protoplast yield, plating efficiency and transient expression of an alien gene

Ethylene release by potato shoots cultured in closed boxes was suppressed by the addition of silver thiosulfate to the culture medium. Shoots cultured in the presence of silver thiosulfate produced appreciably more tissue and the yield of protoplasts per unit tissue mass was vastly increased, resulting in an 8 fold increase of protoplast yield per shoot. Exposure of pricked leaves to macerating enzymes facilitated ethylene generation. Leaves of shoots which were previously cultured in silver thiosulfate containing medium generated much less ethylene than leaves from control shoots and this generation could be further reduced by the addition of acetylsalicylic acid during maceration. The capability of polyethylene glycol treated potato protoplasts to produce microcalli was vastly increased by the addition of silver thiosulfate during exposure of protoplasts to Ca(NO3)2 following the polyethylene glycol treatment. Similarly, when a plasmid (pCAP212) containing an expressible gene for chloramphenicol acetyltransferase was introduced into potato protoplasts through a polyethylene glycol treatment, the transient expression of acetyltransferase was very much increased by the addition of a short incubation of the protoplasts with silver thiosulfate.