Cost-effective in vitro propagation methods for pineapple.

We have developed an efficient and cost-effective method for commercial micropropagation of Smooth Cayenne pineapple. In vitro shoots were used as starting materials, and either longitudinal sections of the shoots or leaf bases were used as the explants to regenerate shoots. When these explants were used, the axillary meristems, which usually remain quiescent during shoot multiplication, were able to form new shoots. Subsequent to the regeneration step, additional multiplication was achieved inside a 10-l Nalgene vessel with shoots immersed in liquid medium for 5-10 min/h (periodic immersion bioreactor, PIB). The shoots were then induced to form roots and transferred to soil. Using the above micropropagation method and the PIB, we produced 6,000-8,000 shoots from two initial shoots in less than 6 months. The clonal fidelity of propagated plants was tested in Costa Rican and Indonesian pineapple farms.

A simple method to enrich an Agrobacterium-transformed population for plants containing only T-DNA sequences.

A simple modification to standard binary vector design has been utilized to enrich an Agrobacterium-transformed population for plants containing only T-DNA sequences. A lethal gene was incorporated into the non-T-DNA portion of a binary vector, along with a screenable marker. The resulting class of vectors is designated as NTL T-DNA vectors (non-T-DNA lethal gene-containing T-DNA vectors). The lethal gene used here is a CaMV 35S-barnase gene with an intron in the coding sequence (barnase-INT); the screenable marker is a pMAS-luciferase gene with an intron in the coding sequence (LUC-int). To evaluate the utility of this vector design, tobacco plants were transformed with either the NTL T-DNA vector or a control vector from which most of the barnase-INT gene was deleted. Populations of 50 transgenic plants were scored for LUC expression. The results indicated a dramatic reduction in the presence of non-T-DNA sequences in the transgenic population using the NTL T-DNA vector. Only one transgenic plant was found to be LUC+ using the NTL vector, compared with 42 of 50 plants using the control vector. Importantly, the efficiency with which transformed tobacco plants was obtained was reduced by no more than 30%. The reduction in LUC+ transgenics was partially reversed when a barstar-expressing tobacco line was transformed, indicating that barnase expression was responsible for the reduced frequency of incorporating non-T-DNA sequences. Similar transformation results were obtained with tomato and grape. The incorporation of a barnase-INT gene outside the left border appears to provide a generally applicable tool for enriching an Agrobacterium-transformed population for plants containing only T-DNA sequences.

Enhanced frequency of transposition of the maize transposable element Activator following excision from T-DNA in Petunia hybrida.

Many of the systems currently employed for heterologous transposon tagging in plants rely on an excision assay to monitor transposon activity. We have used the streptomycin phosphotransferase (SPT) reporter system to assay Ac activity in Petunia hybrida. In other species, such as tobacco or Arabidopsis, excision of Ac from the SPT gene in sporogenous tissue gives rise to streptomycin-resistant seedlings in the following generation. The frequency of fully streptomycin-resistant seedlings in petunia was low (0.4%) but molecular analysis of these indicated that the actual excision frequency may be as low as 0.05%. This indicates that the SPT assay is not a reliable selection criterion for germinal excision in petunia. Extensive molecular screening for reinsertion of Ac was consistent with a low primary transposition frequency (0%-0.6%). In contrast to these findings, the progeny of confirmed germinal transpositions for three independent transformants showed frequent transposition to new sites (9.5%-17.0%). This suggests a high frequency of secondary transposition compared with primary transposition from the T-DNA. Segregation analysis indicates that the high transposition activity is closely associated with transposed copies of Ac. No evidence was found for an altered methylation state for Ac following transposition. The implications of these results for heterologous transposon tagging in petunia are discussed in the context of the reliability of excision reporter systems in general.

Modification of flower color in florist's chrysanthemum: production of a white-flowering variety through molecular genetics.

Chimeric chalcone synthase (CHS) constructs were prepared in both anti-sense and sense orientations, and introduced into the chrysanthemum cultivar Moneymaker, along with a T-DNA vector lacking a CHS construct. For both the anti-sense and sense constructs, the majority of the plants produced pink flowers typical of Moneymaker itself. Of 133 sense and 83 anti-sense transgenic individuals 3 of each set produced fully white or very pale pink flowers. No white-flowering transgenic plants were obtained in control transformations. The white flowers were found to accumulate higher levels of chalcone synthase precursors and to have reduced levels of chalcone synthase message. A small-scale field trial was performed to evaluate the stability of the phenotype throughout a series of vegetative propagation steps and during plant growth. The white-flowering trait was maintained well through vegetative propagation; however, during growth of individual white-flowering plants, some pink color was found in some flowers. At one site 2% of the white-flowering plants produced a few pink flowers; at two other sites, as many as 10-12% of the plants produced pale pink flowers.

Tagging and Cloning of a Petunia Flower Color Gene with the Maize Transposable Element Activator.

We report here the use of the maize transposable element Activator (Ac) to isolate a dicot gene. Ac was introduced into petunia, where it transposed into Ph6, one of several genes that modify anthocyanin pigmentation in flowers by affecting the pH of the corolla. Like other Ac-mutable alleles, the new mutation is unstable and reverts to a functional form in somatic and germinal tissues. The mutant gene was cloned using Ac as a probe, demonstrating the feasibility of heterologous transposon tagging in higher plants. Confirmation that the cloned DNA fragment corresponded to the mutated gene was obtained from an analysis of revertants. In every case examined, reversion to the wild-type phenotype was correlated with restoration of a wild-type-sized DNA fragment. New transposed Acs were detected in many of the revertants. As in maize, the frequency of somatic and germinal excision of Ac from the mutable allele appears to be dependent on genetic background.

Genetic determinants for catabolite induction of antibiotic biosynthesis in Pseudomonas fluorescens HV37a.

Antibiotic biosynthesis is regulated by glucose in Pseudomonas fluorescens HV37a. Fusions between antibiotic biosynthetic operons (afu operons) and the Escherichia coli lac operon were isolated to evaluate the genetic determinants for the regulation of antibiotic biosynthesis. Four afu transcriptional units were defined, afuE, afuR, afuAB, and afuP. The afuE and afuR transcripts were promoted divergently at one locus and were catabolite induced, by 250-fold and 5-fold, respectively; the afuAB and afuP transcriptional units were not linked to the others and were not catabolite induced. Thus, regulation of afuE and afuR operon transcription is apparently the mechanism whereby glucose regulates antibiotic biosynthesis. Catabolite induction of the afuE and afuR transcriptional unit was dependent on the products of the afuA, afuB, and afuP genes. Expression of the afuE transcriptional unit was altered quantitatively in afuE mutants. Apparently the afuE transcriptional unit is regulated, at least in part, by its own gene products. Under inducing conditions, expression of the afuE, afuR, and afuP transcriptional units increased rapidly during a 6-h period.

An efficient mobilizable cosmid vector, pRK7813, and its use in a rapid method for marker exchange in Pseudomonas fluorescens strain HV37a.

We describe the construction and utilization of a new mobilizable cosmid vector. Using this vector, mobilizable libraries of bacterial DNA can be efficiently made without a need for size fractionation of target DNA. The low stability of this vector in Pseudomonas fluorescens makes it useful in a rapid strategy, which is not dependent on plasmid incompatibility, for recombining transposon-induced mutations into the bacterial chromosome.

Multiple antibiotics produced by Pseudomonas fluorescens HV37a and their differential regulation by glucose.

Pseudomonas fluorescens HV37a inhibited growth of the fungus Pythium ultimum on potato dextrose agar (PDA). An antibiotic activity produced under these conditions was fractionated and partially characterized. Extracts prepared from the PDA on which HV37a was grown revealed a single peak of antibiotic activity on thin-layer chromatograms. Similar extracts were prepared from mutants of HV37a. Their analysis indicated that the antibiotic observed in thin-layer chromatograms was responsible for fungal inhibition observed on PDA. The production of the PDA antibiotic required the presence of glucose, whereas two other antibiotic activities were produced only on potato agar without added glucose. Two mutants (denoted AfuIa and AfuIb) previously characterized as deficient in fungal inhibition on PDA showed altered regulation of the production of all three antibiotics in response to glucose. These mutants were also deficient in glucose dehydrogenase. Mutants isolated as deficient in glucose dehydrogenase were also deficient in fungal inhibition and were grouped into two classes on the basis of complementation analysis with an AfuI cosmid. Glucose regulation of antibiotic biosynthesis therefore involves at least two components and requires glucose dehydrogenase.

Molecular cloning of genetic determinants for inhibition of fungal growth by a fluorescent pseudomonad.

Pseudomonas fluorescens HV37a inhibits growth of the fungus Pythium ultimum in vitro. Optimal inhibition is observed on potato dextrose agar, a rich medium. Mutations eliminating fungal inhibition were obtained after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. Mutants were classified by cosynthesis and three groups were distinguished, indicating that a minimum of three genes are required for fungal inhibition. Cosmids that contain wild-type alleles of the genes were identified in an HV37a genomic library by complementation of the respective mutants. This analysis indicated that three distinct genomic regions were required for fungal inhibition. The cosmids containing these loci were mapped by transposon insertion mutagenesis. Two of the cosmids were found to contain at least two genes each. Therefore, at least five genes in HV37a function as determinants of fungal inhibition.

A diffusion assay for detection and quantitation of methyl-esterified proteins on polyacrylamide gels.

The methyl esterification of bacterial and mammalian proteins is a subject of increasing interest and effort. Such studies in intact cells typically involve the use of [methyl-3H]methionine which is taken up and incorporated into S-adenosyl-L-methionine, the methyl donor. The level of methylation, however, is much less than the incorporation of labeled methionine directly into protein. A diffusion assay which distinguishes [3H]methionine from the base-labile [3H]methyl esters is described here. The ester linkage is hydrolyzed at high pH to release [3H]methanol from the sample which diffuses into an adjacent pool of scintillation fluid. The assay is contained in a scintillation vial which can be counted directly.

Replacement and amplification of bacterial genes with sequences altered in vitro.

An efficient method for the replacement of chromosomal DNA by segments altered in vitro has been developed for bacteria. The method requires (i) a recombinant plasmid with a ColE1-like replicon and (ii) a strain defective in DNA polymerase I (polA), which is unable to replicate the plasmid extrachromosomally. This method is of general use since there are a number of suitable vectors and polA strains are available in both Escherichia coli and Salmonella typhimurium, the two most widely studied bacterial species. Using the method, we have constructed two chromosomal deletions in the chemotaxis gene region of S. typhimurium. In addition, plasmid sequences integrated into the chromosome have been amplified up to 30-fold by varying the concentration of ampicillin or tetracycline in the growth medium.