Isolation of deficiencies in the Arabidopsis genome by gamma-irradiation of pollen.

Chromosomal deficiencies are a useful genetic tool in fine-scale genetic mapping and the integration of physical and visible marker genetic maps. Viable overlapping deficiencies may permit gene cloning by subtractive procedures and provide a means of analyzing the functional importance of different chromosomal regions. A method is described for isolation of deficiencies in the Arabidopsis genome which encompass specific loci and other extended chromosomal regions. The technique employs pollen mutagenized by gamma-irradiation to pollinate marker lines homozygous for recessive mutations. Deficiencies at specific loci were detected by screening for marker phenotypes in the F1. Screening for lethal mutations in the F1/F2 confirmed specific deficiencies and revealed other deficiencies that did not overlap the marker loci. Further evidence for such mutations was provided by distorted F2 segregation of the chromosomal markers linked to putative deficiencies. Maintainable (transmissible) and non-transmissible deficiencies were demonstrated by their pattern of inheritance in subsequent generations.

Characterization of gamma irradiation-induced deletion mutations at a selectable locus in Arabidopsis.

Seeds of transgenic Arabidopsis, containing a negatively selectable suicide marker, a 35Stms2 construct introduced as a transgene, were gamma-irradiated at a range of doses from 20-120 krad. Batches of M2 seeds, from M1 plants irradiated at doses of 40, 45 and 60 krad, were screened by germinating them on medium containing NAM under conditions that selectively inhibited growth of plants expressing the tms2 gene product. Nine candidate loss-of-transgene mutants were isolated. The frequency of such mutations (0.0125 to 0.025%) did not vary significantly with irradiation dose or M1 pool size. DNA from the mutants and the parent was hybridized in Southern blots, using probes complementary to various regions of the transgene. All nine mutants were null for both the tms2 coding sequence and the 35S promoter. Six of the nine mutants were null for the entire transgene construct of 9 kbp. DNA from one mutant contained one of the T-DNA borders and gave a hybridization pattern consistent with a deletion at least 5 kbp. The two remaining mutant lines gave identical patterns of hybridization, consistent with a 5.6-kbp internal deletion within the transgene. From the Southern blots, and on the basis of lineage, the nine lines represent the progeny of either seven or eight independent mutations. We have established conditions capable of producing deletion mutations of at least 5 kbp, but without apparently introducing small deletions or rearrangements. Such deletion mutations are ideally suited for cloning by subtractive hybridization, and should also be readily detectable by RFLP analysis, facilitating map-based cloning procedures.

Design and use of synthetic oligonucleotide probes in the cloning of delta-endotoxin genes from Bacillus thuringiensis.

A detailed protocol is described for the design and use of synthetic oligonucleotide probes for screening DNA libraries from Bacillus thuringiensis var. kurstaki (strain HD191) for copies of the gene (tox) encoding the insecticidal delta-endotoxin. Two homologous tox genes were identified in this organism; one of these was located on a 75-kb plasmid and the other on a second large plasmid or the bacterial chromosome. A tox gene was isolated as a 6.5-kb HindIII fragment of B. thuringiensis plasmid DNA.

Emulsified perfluorochemicals for oxygenation of microbial cell cultures?

PFCs and their emulsions may have value for increasing the efficiency of O2-transport to microbial cultures. Therefore, the effects of emulsion components on growth of S. cerevisiae and E. coli have been examined. Viable cell counts revealed that perfluorodecalin or the commercial emulsion, Fluosol-DA 20%, produced no obvious growth-inhibition over 6h. However, incubation of cells with up to 10% of the Pluronic F-68 surfactant reduced absorbance at 600nm. Further experiments to assess the effects of PFC emulsion components on growth and structure of microbial cells are in progress.

Perfluorochemicals for gas transport and improvement of cell cultures.

This paper has considered the effects and potential application of PFCs, their emulsions and emulsion components for regulating growth and metabolic functions of microbial, animal and plant cells in culture. PFCs will help to overcome problems encountered in conventional culture systems (e.g. limited gas supply, mechanical damage), especially where cells are grown to high density. While the commercial potential of PFCs for in vitro systems has not yet been fully exploited, the most exciting areas for future developments are in the culture of animal and plant cell lines of importance in biotechnology and medicine.

Genetics of gamma-irradiation-induced mutations in Arabidopsis thaliana: large chromosomal deletions can be rescued through the fertilization of diploid eggs.

Despite the demonstrated value of chromosomal deletions and deficiencies as tools in plant and animal genome research, in the genetic model plant species Arabidopsis thaliana, such mutations have not been extensively studied. For example, it is not known whether large deletions in different regions of the genome can be tolerated in diploid plants that are heterozygous for such mutations. Similarly the viability or inviability of monosomics has not been examined in detail. To investigate these questions, we have used gamma-irradiated haploid wild-type pollen to pollinate diploid and tetraploid multimarker lines of Arabidopsis. Examination of M1 progenies revealed that chromosome loss mutations and large deletions were induced in the irradiated pollen. Such mutations were eliminated in diploid M1 plants due to dominant lethality but could be rescued in triploid M1 progeny. The use of irradiated pollen and tetraploid marker lines of Arabidopsis is a convenient way of generating deletions and modified chromosomes and provides a genetic tool for deletion mapping and for analysis of chromosomal regions essential for chromosome maintenance.

Transformation of the genomic expression of plant cells.

Agrobacterium-induced transformation of plant cells results from integration of T-DNA of the Ti or Ri plasmids into the genome of susceptible plants. Expression of T-DNA genes induces physiological changes in transformed cells which modify normal plant development to produce proliferations characteristic of crown gall and hairy root diseases. Understanding of the molecular basis of the transformation events associated with these examples of naturally occurring genetic engineering of plant cells, has stimulated efforts to construct vectors for transferring specific genes into plants. Vector construction has progressed from the use of wild-type Ti plasmids, giving phenotypically abnormal regenerated plants, to non-oncogenic plasmids. The range of vectors now available should enable useful foreign genes to be inserted into a range of dicotyledons and monocotyledons without impairing normal plant development.

Direct DNA transfer to plant cells.

A range of somatic cell and molecular techniques are now available to supplement conventional plant breeding. The introduction and expression of foreign DNA has been used to modify basic aspects of physiology and development, to introduce commercially important characteristics such as herbicide and insect resistance into plants and to insert genes suitable as dominant selectable markers for somatic hybridisation. Several techniques for direct DNA delivery are available, ranging from uptake of DNA into isolated protoplasts mediated by chemical procedures or electroporation, to injection and the use of high-velocity particles to introduce DNA into intact tissues. Direct DNA uptake is applicable to both stable and transient gene expression studies and utilises a range of vectors, including those employed for gene cloning. Although the frequency of stable transformation is low, direct DNA uptake is applicable to those plants not amenable to Agrobacterium transformation, particularly monocotyledons.

Interspecific somatic hybrids ofRudbeckia hirta andR. Laciniata (Compositae).

Interspecific somatic hybrid plants betweenRudbeckia hirta cv. Marmalade andR.laciniata cv. Irish Eyes were regenerated following the electro-fusion of mesophyll protoplasts ofR.hirta with callus protoplasts ofR.laciniata. A hybrid selection scheme was based on the fact that plant regeneration, from parental protoplasts ofR.hirta, was via shoot regeneration of callus, and only via rhizogenesis forR.laciniata. The other half of the selection strategy was based on the presence of anthocyanin-pigmented roots; a characteristic of theR.hirta parent only. Somatic hybrids were regenerated, via rhizogenesis, alongside normalR.laciniata but were distinguished by the presence of pigmented roots (a feature ofR.hirta). Hybrid plants had a floral morphology that was intermediate as compared to that of the two parents, with an expected somatic chromosome number of 2n=(2x+4x)=74. Pollen viability though was low. Esterase and peroxidase isozyme profiles confirmed the hybrid nature of the regenerated plants with pigmented roots, whilst chloroplast DNA restriction analysis showed that these hybrids had aR.laciniata chloroplast DNA. This demonstration of somatic hybridisation not only opens up the possibility of incorporating novel traits between such ornamentalCompositae species, but provides a selection strategy based on rhizogenesis as the route to plant regeneration coupled with heritable pigmentation production of roots as a confirmatory hybrid marker.

Roots induced on cucumber cotyledons by the agropine Ri plasmid TR-DNA exhibit the transformed phenotype.

Cucumber explants were transformed by Agrabacterium strains carrying Ri plasmids with functional TL and TR-DNAs, and by strains whose pRi had an intact TR-DNA but a disarmed TL-DNA lacking open reading frames (ORFs) 3 to 9, 10 (rol A), 11 (rol B), 12 (rol C), 13, 14, 15 (rol D), 16 and 17. Roots induced by all strains exhibited extensive root hair formation under axenic conditions, synthesised opines, and contained TR-specific DNA. These results confirm that the TR-DNA of an agropine Ri plasmid is able to elicit the transformed root phenotype in this plant.

Effects of Pluronic F-68 on callus growth and protoplast plating efficiency of Solanum dulcamara.

The effects of the non-ionic surfactant, Pluronic F-68, on the growth of callus and protoplasts from Solanum dulcamara L. have been studied. Growth of callus was stimulated by addition of 0.1% (w/v) commercial grade Pluronic to culture medium, whereas lower concentrations (0.01% w/v) had no corresponding effect. In contrast, higher concentrations (1.0% w/v) of Pluronic inhibited callus growth. The mean plating efficiency of protoplasts grown at different densities (15 days after plating) was increased up to 26% following culture with 0.1% (w/v) Pluronic, while 0.01% (w/v) Pluronic was ineffective. Mean protoplast plating efficiency decreased by up to 32% following culture with 1.0% (w/v) Pluronic.

Production of kanamycin resistant rice tissues following DNA uptake into protoplasts.

Rice protoplasts (Oryza sativa L. v Taipei 309) have been transformed to kanamycin resistance following uptake of pCaMVNEO induced by electroporation, PEG and PEG combined with electroporation. Protoplast-derived colonies selected on medium containing 100 µg/ml of kanamycin expressed NPTII activity, and contained DNA that hybridised to a 1.0 Kb BamHI fragment of pCaMVNEO carrying the NPTII gene. Expression of the transformation frequency in relative terms (number of kanamycin resistant colonies compared to the number of colonies on kanamycin free medium) gave frequencies of 26.1%, 8.5% and 2.9% following electroporation, PEG and PEG with electroporation respectively. In absolute terms (number of kanamycin resistant colonies compared to the number of protoplasts plated) these represent frequencies of 19.9×10(-5), 9.0×10(-5) and 2.7×10(-5) for the three procedures.

Small mitochondrial DNA molecules of wild abortive cytoplasm in rice are not necessarily associated with CMS.

Mitochondrial DNA was isolated from leaf tissue of both the cytoplasmic male sterile line of Indica rice variety V41, which carries wild abortive (WA) cytoplasm, and from the corresponding maintainer line. In addition to the main mitochondrial DNA, four small plasmid-like DNA molecules were detected in both the male sterile and fertile lines. Restriction analysis of total mitochondrial DNA from the male sterile and fertile lines showed DNA fragments unique to each. Our findings suggest that the four small mitochondrial DNA (mtDNA) molecules are conserved when WA cytoplasm is transferred into different nuclear backgrounds. However, there is no simple correlation between the presence/ absence of small mitochondrial DNA molecules and the expression of WA cytoplasmic male sterility (CMS).

Stability of mitochondrial DNA in tissue-cultured cells of rice.

Restriction analysis of mitochondrial (mt) DNA from 3-month-old callus cultures of the cytoplasmic male sterile rice, V41A, which contains S2 or "wild abortive" cytoplasm, and its fertile maintainer, V41B, showed the same BamHI restriction profiles as mtDNA from the corresponding leaf material. Similarly, mtDNA of rice (var. Taipei 309) from leaves, a 2-month-old cell suspension (T3MS2/A), a totipotent suspension (T3MS) and a 19-month-old suspension, which had lost its protoplast regeneration ability (LB3), showed indistinguishable BamHI restriction profiles. However, clear differences in mtDNA restriction profiles were observed between LB3 and a 30-month-old suspension culture of Taipei 309 (LB1), which appeared to reflect substantial changes in the relative abundance of specific DNA sequences. Hybridisation of a maizecoxII gene probe to blots of restricted mtDNA confirmed that, while the relative abundance of certain mtDNA sequences was preserved during long-term tissue culture of rice, major changes in abundance were observed with other sequences.

Fine-scale molecular genetic (RFLP) and physical mapping of a 8.9 cM region on the top arm of Arabidopsis chromosome 5 encompassing the male sterility gene, ms1.

Fine-scale molecular mapping has been conducted using 183 recombinants between the markers lutescens (lu; 17.6 cM) and transparent testa glabra (ttg; 35.5 cM) on the top arm of Arabidopsis thaliana chromosome 5. This region contains a number of genes involved in floral development including Ms1, a gene required for the post-meiotic development of pollen. In homozygous ms1 mutant plants, pollen development is aborted soon after microspore release, regardless of environmental conditions. The ms1 mutation is located at 29.8 +/- 0.8 cM on chromosome 5. Markers have been identified which co-segregate with ms1 and should lie within 39 kb of the gene. The fine-scale map of the lu-ms1-ttg region that has been generated is significantly different from the published integrated map and provides substantially more accurate and higher marker density than the current recombinant inbred map for this region. Using clones derived from four yeast artificial chromosome libraries, a contig has been established between the RFLP markers 4111 and 4556, which encompasses the ms1 gene. This covers a genetic distance of 8.9 cM which corresponds to a physical distance of approximately 1.44 Mb, representing about 1.5-2.0% of the Arabidopsis genome. In this region, 1 cM represents a physical distance of approximately 160 kb.

Transformation of sugarcane protoplasts by direct uptake of a selectable chimaeric gene.

Sugarcane protoplasts were transformed to kanamycin resistance at a frequency of approximately 8 in 10(7) following PEG-induced uptake of Sma1 linearised pABD1 plasmid. DNA-treated protoplasts were cultured in agarose droplets, and protoplast-derived transformants selected on 80 µg ml(-1) kanamycin. Transformed tissues maintained on this level of antibiotic expressed APH(3')II activity, and contained DNA that hybridised to a probe with the APH(3')II gene.

The Arabidopsis MALE STERILITY 2 protein shares similarity with reductases in elongation/condensation complexes.

The Arabidopsis thaliana MALE STERILITY 2 (MS2) gene product is involved in male gametogenesis. The first abnormalities in pollen development of ms2 mutants are seen at the stage in microsporogenesis when microspores are released from tetrads. Expression of the MS2 gene is observed in tapetum of wild-type flowers at, and shortly after, the release of microspores from tetrads. The MS2 promoter controls GUS expression at a comparable stage in the tapetum of transgenic tobacco containing an MS2 promoter-GUS fusion. The occasional pollen grains produced by mutant ms2 plants have very thin pollen walls. They are also sensitive to acetolysis treatment, which is a test for the presence of an exine layer. The MS2 gene product shows sequence similarity to a jojoba protein that converts wax fatty acids to fatty alcohols. A possible function of the MS2 protein as a fatty acyl reductase in the formation of pollen wall substances is discussed.

Transgenic rice plants produced by electroporation-mediated plasmid uptake into protoplasts.

Transgenic rice plants have been regenerated by somatic embryogenesis from cell suspension derived protoplasts electroporated with plasmid carrying the NPTII gene under the control of the 35S promoter from cauliflower mosaic virus. Heat shock of protoplasts prior to electroporation maximised the throughput of kanamycin resistant colonies. Omission of kanamycin from the medium for plant regeneration was essential for the recovery of transgenic rice plants carrying the NPTII gene. This report of the production of kanamycin resistant transgenic rice plants establishes the use of protoplasts for rice genetic engineering.

Expression of a chimaeric kanamycin resistance gene introduced into the wild soybeanGlycine canescens using a cointegrate Ri plasmid vector.

Seedling hypocotyl explants ofGlycine canescens were inoculated withAgrobacterium rhizogenes carrying a chimaeric NPTII gene cointegrated into the TL-DNA of pRiA4. Transformed roots produced shoots on B5 based medium with 10.0 mgl(-1) BAP, 0.05 mgl(-1) IBA and 50 µgml(-1) kanamycin. Cultured roots and regenerated plants expressed NPTII enzyme activity which was correlated with the presence of Ri TL-DNA and the structural sequence of the NPTII gene.