Two unlinked T-DNAs can transform the same tobacco plant cell and segregate in the F1 generation

Summary The 200 kb Agrobacterium Ti-plasmid pTiT37 carries a 25 kb segment of T-DNA which it transfers to plant cells during crown-gall tumorigenesis. We have previously engineered into this T-DNA a pBR322-derived cloning vector which enabled us to rescue-clone full length T-DNA from the Ti-plasmid into a 36 kb MINI-Ti plasmid. We report here the deletion of oncogenes from MINI-Ti to produce Micro-Ti containing the nopaline synthase gene and the ampicillin resistance gene and origin of replication of pBR322, flanked by left and right T-DNA borders. Micro-Ti was recloned into the wide host range plasmid pRK290 and transformed into an A. tumefaciens strain carrying a helper plasmid that could supply Virulence (VIR) genes in trans. Using the octopine Ti-plasmid pTiB6-806 as a helper, transformed tobacco cells were obtained which produced both nopaline and octopine. Two cloned cell lines producing both opines were found to be hormone dependent and to produce fertile tobacco plants. We selfed one of these plants and found that the two opine markers segregated in the F1 progeny in a Mendelian fashion. This showed that the T-DNAs were not linked in the transformed plant genome. Southern blot analysis of the genomic DNA from the regenerated plant showed that only part of the (oncogenic) octopine T-DNA was present indicating that it had suffered a deletion in the auxin producing locus (tms region). Presence of the cytokinin autonomy locus presumably accounts for the abnormal rooting behavior of the F1 progeny seedlings containing this T-DNA.Abbreviations NAA Naphtalene acetic acid - IAA Indole-3-acetic acid - BA 6-benzylaminopurine - pCPA para-chlorophenoxyacetic acid Part of this work was presented for her doctoral thesis by A. JdF at the National Institute of Agronomy of Paris-Grignon, January 1983     

Transformation of plant protoplasts with DNA: cotransformation of non-selected calf thymus carrier DNA and meiotic segregation of transforming DNA sequences

Summary With the DNA transformation procedure developed in our laboratory (13) several transformed tobacco SR1 tissues were obtained which, apart from selected and non-selected pTi sequences (T⁺), also had acquired non-selected calf thymus carrier DNA sequences (C⁺), being integrated in their nuclear genomes. From one such tissue (cNT4), with a shooty crown gall phenotype and expressing mannopine synthesis activity (Mas⁺), shoots were grafted and mature, flowering plants (gNT4) were obtained. After cross pollination with wild type SR1 tobacco pollen 49% of the seedlings obtained, had the maternal NT4-like crown gall phenotype and 51% showed wild type (SR1) features. The mannopine locus segregated independently from the locus determining the crown gall phenotype. When screened for integrated (transforming) foreign DNA sequences 97% of the NT4-like seedlings turned out to be C⁺T⁺. Most of the SR1-like seedlings, having a wild type tobacco morphology, proved to be transformed as well: roughly a 1:1:1:1 ratio as found for C⁺T⁺:C^-T⁺: C⁺T:C T SR1-like seedlings. Based on the segregation of transforming sequences during meiosis a model is presented showing the integration of these sequences in three different host chromosomes.     

Production and genetic analysis of partial hybrids in intertribal crosses between Brassica species (B. rapa, B. napus) and Capsella bursa-pastoris

Capsella bursa-pastoris (L.) Medic (2n = 4x = 32) is a natural double-low (erucic acid < 1%, glucosinolates < 30 micromol/g) germplasm and shows high degree of resistance to Sclerotinia sclerotiorum. Hybridizations were carried out between two Brassica species viz. B. rapa (2n = 20) and B. napus (2n = 38) as female and C. bursa-pastoris as male parent to introduce these desirable traits into cultivated Brassica species. Majority of F(1) plants resembled female parents in morphology and only a few expressed some characters of male parent, including the white petals. Based on cytological observation of somatic cells, the F(1) plants were classified into five types: two types from the cross with B. rapa, type I had 2n = 27-29; type II had 2n = 20; three types from the crosses with B. napus, type III was haploids with 2n = 19; type IV had 2n = 29; type V had 2n = 38. One to two chromosomes of C. bursa-pastoris were detected in pollen mother cells (PMCs) of type I plant by genomic in situ hybridization (GISH), together with chromosomal segments in ovary cells and PMCs of some F1 plants. Amplified fragment length polymorphism (AFLP) bands specific for the male parent, novel for two parents and absent bands in Brassica parents were generated in majority of F1 plants, even in Brassica-types and haploids, indicating the introgressions at various levels from C. bursa-pastoris and genomic alterations following hybridization. Some Brassica-type progeny plants had reduced contents of erucic acid and glucosinolates associated with improved resistance to S. sclerotiorum. The cytological and molecular mechanisms behind these results are discussed.     

A Preliminary Genetic Linkage Map of the Pacific Abalone Haliotis discus hannai Ino

Preliminary genetic linkage maps were constructed for the Pacific abalone (Haliotis discus hannai Ino) using amplified fragment length polymorphism (AFLP), randomly amplified polymorphic DNA (RAPD), and microsatellite markers segregating in a F₁ family. Nine microsatellite loci, 41 RAPD, and 2688 AFLP markers were genotyped in the parents and 86 progeny of the mapping family. Among the 2738 markers, 384 (including 365 AFLP markers, 10 RAPD markers, and 9 microsatellite loci) were polymorphic and segregated in one or both parents: 241 in the female and 146 in the male. The majority of these markers, 232 in the female and 134 in the male, segregated according to the expected 1:1 Mendelian ratio (α = 0.05). Two genetic linkage maps were constructed using markers segregating in the female or the male parent. The female framework map consisted of 119 markers in 22 linkage groups, covering 1773.6 cM with an average intermarker space of 18.3 cM. The male framework map contained 94 markers in 19 linkage groups, spanning 1365.9 cM with an average intermarker space of 18.2 cM. The sex determination locus was mapped to the male map but not to the female map, suggesting a XY-male determination mechanism. Distorted markers showing excess of homozygotes were mapped in clusters, probably because of their linkage to a gene that is incompatible between two parental populations.     

Agrobacterium-mediated transformation of Asparagus officinalis L.: molecular and genetic analysis of transgenic plants

Four long-term embryogenic lines of Asparagus officinalis were co-cultured with the hypervirulent Agrobacterium tumefaciens strain AGL1Gin carrying a uidA gene and an nptII gene. 233 embryogenic lines showing kanamycin resistance and -glucuronidase (GUS) activity were obtained. Transformation frequencies ranged from 0.8 to 12.8 transformants per gram of inoculated somatic embryos, depending on the line. Southern analysis showed that usually 1 to 4 T-DNA copies were integrated. Regenerated plants generally exhibited the same insertion pattern as the corresponding transformed embryogenic line. T₁ progeny were obtained from crosses between 6 transformed plants containing 3 or 4 T-DNA copies and untransformed plants. They were analysed for GUS activity and kanamycin resistance. In three progenies, Mendelian 1:1 segregations were observed, corresponding to one functional locus in the parent transgenic plants. Southern analysis confirmed that T-DNA copies were inserted at the same locus. Non-Mendelian segregations were observed in the other three progenies. T₂ progeny also exhibited non-Mendelian segregations. Southern analysis showed that GUS-negative and kanamycin-sensitive plants did not contain any T-DNA, and therefore inactivation of transgene expression could not be responsible for the abnormal segregations.     

Locations and stability of Agrobacterium-mediated T-DNA insertions in the Lycopersicon genome

Summary The genomic distribution and genetic behavior of DNA sequences introduced into the tomato genome by Agrobacterium tumefaciens were investigated in the backcross progeny of 10 transformed Lycopersicon esculentum x L. pennellii hybrids. All transformants were found to represent single locus insertions based on the co-segregation of restriction fragments corresponding to the T-DNA left and right border sequences in the backcross progeny. Isozyme and restriction fragment length polymorphism (RFLP) markers were used to test linkage relationships of the insertion in each backcross family. The T-DNA inserts in 9 of the 10 transformants were mapped in relation to one or more of these markers, and each mapped to a different chromosomal location. Because only one insertion did not show linkage with the markers employed, it must be located somewhere other than the genomic regions covered by the markers assayed. We conclude that Agrobacterium-mediated insertion in the Lycopersicon genome appears to be random at the chromosomal level. No discrepancies were found between the T-DNA genotype and the nopaline phenotype in the 322 backcross progeny of the nopaline positive transformants. Backcross progeny of two nopaline negative transformants showed incomplete correspondence between the T-DNA genotype and the kanamycin resistance phenotype. No alteration of T-DNA was observed in progeny showing a discrepancy between T-DNA and kanamycin resistance. However, two kanamycin resistant progeny plants of one of these two transformants possessed altered T-DNA restriction patterns, indicating genetic instability of the T-DNA in this transformant.Journal article no. 1223 of the New Mexico Agricultural Experiment Station     

Neoplastic progression in crown gall in tobacco without elevated auxin levels

We have isolated two stable variants from a crown-gall teratoma tissue of tobacco (Nicotiana tabacum L.) transformed by Agrobacterium tumefaciens strain A66, a mutant of the virulent A6 strain containing an insertion sequence in the tumor-inducing (Ti) plasmid at the locus coding for auxin biosynthesis. Normally tobacco cells transformed by strain A66 spontaneously form shoots in culture and will not grow on hormone-free medium unless shoots develop. The variant tissue lines, isolated from the teratoma tissue after prolonged culture in the dark, grew as friable and unorganized tissues on hormone-free growth medium. Growth of the variants was more sensitive to auxin feeding than growth of the parental teratoma line, and the auxin dose-response curves of the variant lines were similar to those obtained with A6-transformed tobacco cells. Southern blot analysis of DNA from the parental teratoma line and one of the variants showed no differences in copy number or organization of the oncogenic DNA sequence (T-DNA) transferred from the bacterium, indicating that the variant phenotype did not result from reversion of the A66 mutation. Radio-immunoassay analysis showed similar levels of indole-3-acetic acid (IAA) in the variants and parental teratoma line (3–50 and 38–42 pmol·(gFW)^-1, respectively), whereas an A6-transformed cell line contained much higher IAA levels (150–1200 pmol·(g FW)^-1). Low levels of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid in the variants and the parental teratoma line (<5 nmol·(g FW)^-1) as compared with that found in the A6-transformed line (>100 nmol· (g FW)^-1) provided additional, indirect evidence for low auxin levels in the variant lines. These results indicate that crown-gall teratoma tissues of tobacco may switch to the unorganized, auxin-sensitive phenotype without an increase in auxin content.Abbreviations IAA indole-3-acetic acid - kb kilobase - NAA -naphthalene acetic acid - NAM -naphthaleneacetamide - T-DNA DNA transferred from the Ti plasmid to the plant - T_L-DNA the left transferred region of pTiA6 containing the T-DNA oncogenes     

送春与多花兰种间杂交后代的ISSR分析

该文使用简单重复序列间( ISSR)分子标记,对送春与多花兰种间杂交后代进行了研究。结果表明：从80个ISSR引物中筛选出14个扩增效果稳定的ISSR引物,对两亲本和59个F1代个体进行了ISSR扩增,得到107个扩增位点,扩增的片段大小位于90~2100 bp之间,平均每个引物扩增7.64条条带,得到11种类型的带。 ISSR标记在送春×多花兰的F1代中表现出一定的多态性,分离频率为44.86％,分离位点有83.33％符合孟德尔1︰1或3︰1的分离规律,产生偏孟德尔分离的位点占12.50％,余下的4.17％属于特殊分离带型。可能导致后代变异的位点为偏孟德尔分离的6条带、缺失的8条带或新生成的2条带。聚类图中父本和母本与F1代个体间的遗传距离较远,59个杂交后代先聚集成一组,再同母本相聚为一组,最后才同父本聚在一起,59个杂种均偏母本型。送春与多花兰的杂交后代在植株形态、染色体、遗传物质方面都具备双亲特点,61个个体间的ISSR分子量标记结果和植株形态学特征都说明,59个F1代杂种包含送春和多花兰的遗传特性是真杂种;F1代杂种既有双亲的互补特征带,又有双亲的重组片断即产生新的特异带,这说明送春与多花兰的杂交后代具有遗传变异的特点。该研究结果可以有效地对杂交后代进行定向选择,为兰花的杂交育种提供了分子依据。     

Construction of a genetic linkage map in <Emphasis Type="Italic">Fenneropenaeus chinensis</Emphasis> (Osbeck) using RAPD and SSR markers

The primary genetic linkage maps of Fenneropenaeus chinensis (Osbeck) were constructed by using the “two-way pseudo-testcross” strategy with RAPD and SSR markers. Parents and F1 progeny were used as segregating populations. Sixty-one RAPD primers and 20 pairs of SSR primers were screened from 460 RAPD primers and 44 pairs of SSR primers. These primers were used to analyze the parents and 82 progeny of the mapping family. About 146 primers (128 RAPDs, 18 microsatellites) in the female and 127 primers (109 RAPDs, 18 microsatellites) in the male were segregating markers. The female linkage map included eight linkage groups, nine triplets and 14 doublets, spanning 1,173 cM with the average marker density of 11.28 cM, and the observed coverage was 59.36%. The male linkage map included 10 linkage groups, 12 triplets and seven doublets, spanning 1,144.6 cM with the average marker density of 12.05 cM, and the observed coverage was 62.01%. The construction of the F. chinensis genetic linkage maps here opened a new prospect for marker-assisted selection program, comparative genomics and quantitative trait loci (QTL) gene location and cloning.     

AFLP Linkage Map of the OomycetePhytophthora infestans

Here we present the first comprehensive genetic linkage map of the heterothallic oomycetous plant pathogenPhytophthora infestans.The map is based on polymorphic DNA markers generated by the DNA fingerprinting technique AFLP (Voset al.,1995,Nucleic Acids Res.23:4407–4414). AFLP fingerprints were made from single zoospore progeny and 73 F1 progeny from two field isolates ofP. infestans.The parental isolates appeared to be homokaryotic and diploid, their AFLP patterns were mitotically stable, and segregation ratios in the F1 progeny were largely Mendelian. In addition to 183 AFLP markers, 7 RFLP markers and the mating type locus were mapped. The linkage map comprises 10 major and 7 minor linkage groups covering a total of 827 cM. The major linkage groups are composed of markers derived from both parents, whereas the minor linkage groups contain markers from either the A1 or the A2 mating type parent. Non-Mendelian segregation ratios were found for the mating type locus and for 13 AFLP markers, all of which are located on the same linkage group as the mating type locus.     

Hormonal regulation of zeatin-riboside accumulation by cultured tobacco cells

Auxin (11 M -naphthaleneacetic acid) and cytokinin (1.4 M kinetin) regulate cytokinin accumulation by cytokinin-requiring (C^-) and cytokinin-autotrophic (C⁺) lines of Havana 425 tobacco (Nicotiana tabacum L.) tissues. No trans-zeatin riboside (ZR) (<0.5 pmol·g^-1 fresh weight) was detected in six C^- and nine C⁺ lines grown for 14 d on auxin + cytokinin and auxin medium, respectively. C⁺ lines, but not C^- lines accumulated ZR (1.9–5.1 pmol·g^-1 fresh weight) when incubated on hormone-free medium but both lines accumulated ZR when incubated on kinetin medium. Therefore, it appears that kinetin treatment can induce ZR accumulation and that this accumulation is blocked by auxin treatment. Similar effects were obtained with some lines of cells autotrophic for both auxin and cytokinin. Tobacco plants carrying the dominant Habituated leaf-1 allele (Hl-1) differ from wild-type plants in that leaf-derived tissues in culture exhibit a C⁺ phenotype. No differences in ZR content were found in C⁺ leaf tissues from Hl-1/Hl-1 plants and C⁺ tissues that arise epigenetically in wild-type plants. This indicates that the H-1 allele does not act to induce overproduction of ZR. The Hl-1 allele is known to have oncogenic functions similar to the isopentenyl transferase (ipt) locus of the Ti plasmid. Although Hl-1/Hl-1 cells transformed with Ti plasmids defective at the ipt locus are tumorigenic and hormone-autotrophic in culture, they contain low levels of ZR typical of non-transformed Hl-1/Hl-1 cells. Therefore, the high levels of ZR characteristics of cells transformed with wild-type Ti plasmids are not necessary for expression of the tumor phenotype.Abbreviations C^- cytokinin-requiring phenotype - C⁺ cytokinin-autotrophic phenotype - Hl-1 habituated leaf-1 locus - IPA isopentenyladenosine - ipt isopentenyltransferase gene - ZR trans-zeatin riboside     

Insertional mutagenesis in Arabidopsis thaliana: isolation of a T-DNA-linked mutation that alters leaf morphology

Summary We investigated the potential of the Agrobacterium tumefaciens T-DNA as an insertional mutagen in Arabidopsis thaliana. Arabidopsis lines transformed with different T-DNA vectors were generated using a leaf disc infection procedure adapted for efficient selection on either kanamycin or hygromycin medium. A standardized screening procedure was developed for the detection of recessive mutations in T2 populations of regenerated and/or transformed lines. Recessive mutations originating from the tissue culture procedure occurred at a low frequency — between 2% and 5%. Within 110 transformed lines that contained a total of about 150 T-DNA inserts, one recessive mutation, named pfl, cosegregated with a specific T-DNA copy. This pfl mutation mainly affected the morphology of the first seedling leaves under normal growth conditions and was mapped to chromosome 1. No recombination between the pfl locus and the kanamycin resistance marker on the T-DNA was detected when screening F2 and F3 populations of a mutant crossed to the wild type. The maximal genetic distance between the pfl locus and the kanamycin resistance gene, determined as 0.4±0.4 cMorgan, strongly suggests that the pfl mutation is induced by the insertion of the T-DNA. Our finding of one T-DNA-linked recessive mutation in 110 transgenic lines indicates that T-DNA can be used for mutagenization of the Arabidopsis genome under tissue culture conditions.     

Responses ofArabidopsis roots to auxin studied with high temporal resolution: Comparison of wild type and auxin-response mutants

We modified a video digitizer system to allow short-term high-resolution measurements of root elongation in intact seedlings ofArabidopsis thaliana (L.) Heynh. We used the system to measure the kinetics of promotion and inhibition of root elongation by applied auxin and to determine the dose-response relationship for auxin action on elongation in roots of wild-type seedlings and seedlings of mutants (axr1,aux1, andaxr2) with altered auxin responsiveness. Roots of the mutants showed less inhibition in the presence of inhibitory concentrations of auxin than did roots of the wild type. The latent period preceding the change in elongation rate after auxin application was the same foraxr1 andaxr2 as for the wild type whereas the latent period foraux1 was about twice as long as for the wild type. Low concentrations (ca. 10^–11 M) of auxin induced substantial promotion of root elongation in the wild type and inaxr2.We thank Linda Young and Roger Hangarter for helping to develop the system for mountingArabidopsis seedlings and Wendy Hankie, Julia Hufford, and Ruperto Villella for doing some of the experiments. We thank Roger Hangarter for valuable discussions of the data. This work was supported by National Science Foundation Grant No. DCB-9105807 and by National Aeronautics and Space Administration Grant No. NAG10-0084     

In planta transformation of Arabidopsis thaliana

Transformants of Arabidopsis thaliana can be generated without using tissue culture techniques by cutting primary and secondary inflorescence shoots at their bases and inoculating the wound sites with Agrobacterium tumefaciens suspensions. After three successive inoculations, treated plants are grown to maturity, harvested and the progeny screened for transformants on a selective medium. We have investigated the reproducibility and the overall efficiency of this simple in planta transformation procedure. In addition, we determined the T-DNA copy number and inheritance in the transformants and examined whether transformed progeny recovered from the same Agrobacterium-treated plant represent one or several independent transformation events. Our results indicate that in planta transformation is very reproducible and yields stably transformed seeds in 7–8 weeks. Since it does not employ tissue culture, the in planta procedure may be particularly valuable for transformation of A. thaliana ecotypes and mutants recalcitrant to in vitro regeneration. The transformation frequency was variable and was not affected by lower growth temperature, shorter photoperiod or transformation vector. The majority of treated plants gave rise to only one transformant, but up to nine siblings were obtained from a single parental plant. Molecular analysis suggested that some of the siblings originated from a single transformed cell, while others were descended from multiple, independently transformed germ-line cells. More than 90% of the transformed progeny exhibited Mendelian segregation patterns of NPTII and GUS reporter genes. Of those, 60% contained one functional insert, 16% had two T-DNA inserts and 15% segregated for T-DNA inserts at more than two unlinked loci. The remaining transformants displayed non-Mendelian segregation ratios with a very high proportion of sensitive plants among the progeny. The small numbers of transformants recovered from individual T₁ plants and the fact that none of the T₂ progeny were homozygous for a specific T-DNA insert suggest that transformation occurs late in floral development.National Research Council of Canada Publication No. 38003     

Partial hybridization in wide crosses between cultivated sunflower and the perennial Helianthus species H. mollis and H. orgyalis

To obtain introgressed sunflower lines with improved disease resistance, interspecific crosses were performed with foreign perennial species. We report on several unusual features displayed by these hybrid plants. The methods used to produce the kernels affected yield and genotypes of progeny. Phenotypic traits and DNA markers were investigated in 97 plants derived from cross-pollination between annual diploid cultivated sunflower (Helianthus annuus) and the perennial diploid species H. mollis or H. orgyalis, and the reverse reciprocal crosses. The level of hybridization in progeny was determined using RAPD and RFLP markers. Hybridization was performed by leaving embryos to develop normally on the head (classical crossing) or using embryo rescue. All observed plants derived from H. mollis were diploid (2n = 34). Phenotypes were predominantly similar to the female when cultivated sunflower was the female parent. Progeny from crosses using a wild species as the female parent resembled that parent. Thus, reciprocal crosses led to different progeny. F1 sister progeny shared different sets of molecular markers representing a few of those of the wild species used as the pollen donor. Our results indicate mechanisms leading to the unusual event of partial hybridization. Possible mechanisms behind these unusual events and their possible impact on evolution are discussed.     

Identification of AFLP molecular markers for resistance against Melampsora larici-populina in Populus

We have identified AFLP markers tightly linked to the locus conferring resistance to the leaf rust Melampsora larici-populina in Populus. The study was carried out using a hybrid progeny derived from an inter-specific, controlled cross between a resistant Populus deltoides female and a susceptible P. nigra male. The segregation ratio of resistant to susceptible plants suggested that a single, dominant locus defined this resistance. This locus, which we have designated Melampsora resistance (Mer), confers resistance against E1, E2, and E3, three different races of Melampsora larici-populina. In order to identify molecular markers linked to the Mer locus we decided to combine two different techniques: (1) the high-density marker technology, AFLP, which allows the analysis of thousands of markers in a relatively short time, and (2) the Bulked Segregant Analysis (BSA), a method which facilitates the identification of markers that are tightly linked to the locus of interest. We analyzed approximately 11,500 selectively amplified DNA fragments using 144 primer combinations and identified three markers tightly linked to the Mer locus. The markers can be useful in current breeding programs and are the basis for future cloning of the resistance gene.     

Chromosomal localization of foreign genes in Petunia hybrida

Summary A F1 hybrid of Petunia hybrida, heterozygous for at least one marker on each of the seven chromosomes, was transformed with a modified strain of Agrobacterium tumefaciens in which the phytohormone biosynthetic genes in the transferred DNA (T-DNA) were replaced with a NOS/NPTII/NOS chimeric gene and a wildtype nopaline synthase (NOS) gene. The chimeric gene, which confers kanamycin resistance, was used as selectable marker during the transformation process and the NOS gene was used as a scorable marker in the genetic studies. After plants had been regenerated from the transformed tissues, the transgenic plants that expressed both of these markers were backcrossed to the parental lines. The offspring were examined for the segregation of the NOS gene and the Petunia markers. Genetic mapping was thus accomplished in a single generation.By Southern hybridization analysis we confirmed the presence of the expected T-DNA fragments in the transformed plants. Four out of the six plants presented here, had just one monomeric T-DNA insertion. The sizes of the plant/T-DNA junction fragments suggest that the integration occurred in different sites of the Petunia genome. One transformant gave a more complicated hybridization pattern and possibly has two T-DNA inserts. Another transgenic plant was earlier reported (Fraley et al. 1985) to have two, possibly tandemly repeated T-DNAs.Data is presented on the genetic localization of the T-DNA inserts in six independently obtained transgenic plants. The T-DNA inserts in three plants were mapped to chromosome I. However, the distances between the NOS gene and the marker gene on this chromosome were significantly different. In another transgenic plant the NOS gene was coinherited with the marker on chromosome IV. Two other transgenic plants have the T-DNA insert on chromosome III. A three point cross enabled us to determine that both plants have the NOS gene distally located from the peroxidaseA (prxA) marker and both plants showed about 18% recombination. However, Southern hybridization analysis shows that the sizes of the plant/T-DNA junction fragments in these transgenic plants are different, thus suggesting that the integrations occurred in different sites.     

A study of morphology,cytology and sterility in interspecific hybrids and amphidiploids of Nicotiana knightiana X N. umbratica

Summary Interspecific hybrids and amphidiploids of Nicotiana knightiana Goodspeed (n= 12)x N. umbratica Burbidge (n = 23) resembled either parent in some characters and were intermediate in other characters. The F₁ hybrids (2n = 35) showed mostly univalents during meiosis, while the amphidiploids (2n = 70) formed bivalents almost regularly. The former were completely sterile and the latter fully male fertile but predominantly female sterile. This female sterility was due to disintegration of the embryo sacs leading to collapsed ovules. The few fertile ovules, however, showed normal development of embryo sac and embryo. The occurrence of fertile and sterile ovules was believed to be due to segregation of the genes governing sterility.     

Identification and Mapping of Amplified Fragment Length Polymorphism Markers Linked to Shell Color in Bay Scallop, Argopecten irradians irradians (Lamarck, 1819)

Amplified fragment length polymorphisms (AFLP) were used to study the inheritance of shell color in Argopecten irradians. Two scallops, one with orange and the other with white shells, were used as parents to produce four F₁ families by selfing and outcrossing. Eighty-eight progeny, 37 orange and 51 white, were randomly selected from one of the families for segregation and mapping analysis with AFLP and microsatellite markers. Twenty-five AFLP primer pairs were screened, yielding 1138 fragments, among which 148 (13.0%) were polymorphic in two parents and segregated in progeny. Six AFLP markers showed significant (P < 0.05) association with shell color. All six loci were mapped to one linkage group. One of the markers, F1f335, is completely linked to the gene for orange shell, which we designated as Orange1, without any recombination in the progeny we sampled. The marker was amplified in the orange parent and all orange progeny, but absent in the white parent and all the white progeny. The close linkage between F1f335 and Orange1 was validated using bulk segregation analysis in two natural populations, and all our data indicate that F1f335 is specific for the shell color gene, Orange1. The genomic mapping of a shell color gene in bay scallop improves our understanding of shell color inheritance and may contribute to the breeding of molluscs with desired shell colors.     

Agrobacterium-mediated transformation of germinating seeds of Arabidopsis thaliana: A non-tissue culture approach

Summary Germinating seeds of Arabidopsis thaliana were cocultivated with an Agrobacterium tumefaciens strain (C58Clrif) carrying the pGV3850:pAK1003 Ti plasmid. This Ti plasmid contains the neomycin phosphotransferase II gene (NPT II) which confers resistance to kanamycin and G418. Seeds (T1 generation) imbibed for 12 h before a 24 h exposure to Agrobacterium gave rise to the highest number of transformed progeny (T2 generation). Over 200 kanamycin-resistant T2 seedlings were isolated. Some of the T2 seedlings and T3 families were characterized for genetic segregation of functional NPT II gene(s), NPT II activity, and the presence of T-DNA inserts (Southern analysis). Ninety percent of the T2 individuals transmitted the resistance factor to the T3 families in a Mendelian fashion. Of the T3 families segregating in a Mendelian fashion (n=111), 62% segregated for one functional insert, 29% for two unlinked or linked functional inserts, 5% for three unlinked inserts, 1% for four unlinked inserts, whereas 3% appeared to be homozygous for the insert(s). The 13 families that did not exhibit Mendelian segregation ratios fell into 2 classes, both of which had a deficiency of kanamycin-resistant seedlings. In the Group I T3 families (n=6) only 0%–2% of the seedlings were resistant to kanamycin (100 mg/l), whereas in the Group II families (n=7) 8%–63% of the seedlings were resistant. All of the kanamycin-resistant plants that were tested were found to possess NPT II activity. Southern analysis revealed that all of the resistant plants contained at least one copy of the T-DNA and that the majority of the plants had multiple inserts. Explants from kanamycin-resistant plants survived and formed callus when cultured on callus-inducing medium containg G418.