Agrobacterium tumefaciens-mediated beta-glucuronidase (GUS) gene expression in lentil (Lens culinaris Medik.) tissues.

Lentil (Lens culinaris Medik.) shoot apex, epicotyl, and root expiants were capable of expressing an intron-containing beta-glucuronidase (GUS) gene after inoculation with the disarmed Agrobacterium strain GV2260:p35SGUSINT. Expression occurred at all wound sites on these expiants except at the end of the root expiants proximal to the cotyledonary node. GUS expression was detected using both histochemical and fluorescence assays and was stable for at least nine days after inoculation for epicotyl and root expiants, and for at least seventeen days for shoot apices. Non-inoculated controls, or controls inoculated with an Agrobacterium strain lacking the GUS gene, did not produce any background blue staining in the histochemical assay. Expression levels for all lentil expiants were substantially lower than for comparable flax (Linum usitatissimum L.) expiants which served as a positive control.

Characterization of transgenic sulfonylurea-resistant flax (Linum usitatissimum).

Fourteen transgenic flax (Linum usitatissimum) lines, carrying a mutant Arabidopsis acetolactate synthase (ALS) gene selected for resistance to chlorsulfuron, were characterized for resistance to two sulfonylurea herbicides. Progeny of 10 of the 14 lines segregated in a ratio of 3 resistant to 1 susceptible, indicating a single insertion. Progeny of 1 line segregated in a 15∶1 ratio, indicating two insertions of the ALS gene at independent loci. Progeny from 3 lines did not segregate in a Mendelian fashion and were likely the products of chimeric shoots. Resistance to chlorsulfuron was stably inherited in all lines. At the enzyme level, the transgenic lines were 2.5 to more than 60 times more resistant to chlorsulfuron than the parental lines. The transgenic lines were 25-260 times more resistant to chlorsulfuron than the parental lines in root growth experiments and demonstrated resistance when grown in soil treated with 20 g ha(-1) chlorsulfuron. The lines demonstrated less resistance to metsulfuron methyl; in root growth experiments, the transgenic lines were only 1.6-4.8 times more resistant to metsulfuron methyl than the parental lines. Resistance was demonstrated in the field at half (2.25 g ha(-1)) and full (4.5 g ha(-1)) rates of metsulfuron methyl.

Characterization of an Escherichia coli gene encoding betaine aldehyde dehydrogenase (BADH): structural similarity to mammalian ALDHs and a plant BADH.

An open reading frame of 1476 nucleotides, cloned from a region of the Escherichia coli genome encoding betaine biosynthesis functions, was shown to encode a betaine aldehyde dehydrogenase (BADH; EC 1.2.1.8). Either of two adjacent codons (5'-GTGATG) could function as a start codon, producing a presumptive polypeptide of 491 or 490 amino acids. The deduced primary structure of the E. coli BADH showed 39-43% positional identity, over its entire length, to aldehyde dehydrogenases (ALDH: EC 1.2.1.3) of mammalian origin. This similarity increased to 75-77% when conservative aa substitutions were also taken into consideration. Spinach BADH was also similar to the bacterial BADH, showing 38% identity and 80% overall similarity. Other homologs included a fungal and a putative bacterial ALDH. Although E. coli BADH was specific for the substrate, betaine aldehyde, it showed the highest levels of similarity to the prototype human ALDH-2. Only one gap in each sequence had to be introduced for optimal alignment. The conservation between E. coli BADH and the ALDHs was also evident in the predicted secondary structures and hydrophilicity profiles of the polypeptides, suggesting a similarity in the overall folding patterns of ALDH and BADH. These observations suggest a common ancestry for BADH and ALDH, preceding prokaryote-eukaryote divergence.

Crown gall transformation of lentil (Lens culinaris Medik.) with virulent strains of Agrobacterium tumefaciens.

Four diverse strains of Agrobacterium tumefaciens (C58, Ach5, GV3111, and A281) were capable of inducing tumors at a high frequency on inoculated stems of lentil (Lens culinaris Medik. cultivar Laird) in vivo, and on excised shoot apices in vitro. GV3111 and Ach5 produced the largest and heaviest tumors in vivo, while A281 produced the heaviest tumors in vitro. Tumor formation and opine production are indicative of plant cell transformation and tumors produced appropriate opines: nopaline (C58), octopine (Ach5 and GV3111), and agropine and mannopine (A281). Southern analysis of DNA from a tumor line produced by strain C58 showed that a T-DNA fragment had been transferred into the lentil genome.

Patterns of transformation intensity on flax hypocotyls inoculated with Agrobacterium tumefaciens.

Cellular transformation intensities on flax (Linum usitatissimum) hypocotyl explants using disarmed Agrobacterium tumefaciens were investigated through various preculture durations, cocultivation durations and removal of epidermis. The expression of an intron-containing ß-glucuronidase (GUS) gene driven by CaMV 35S promoter served as a reporter for determination of transformed tissues on hypocotyls. The binary plasmid p35SGUSINT in octopine-type Agrobacterium strain GV2260 was used as the vector system. A prolonged cocultivation duration (5-7 days) resulted in a much higher transformation staining intensity (frequency * tissue area) than 2- or 3-day-cocultivation duration on hypocotyls variously precultured prior to inoculation. A high staining intensity on the two cut ends was obtained from nonprecultured hypocotyls. A reduction in intensity on the upper cut end of hypocotyls was observed with preculture times greater than 6 days. Peeled hypocotyls with a post-peeling preculture of 2 or 3 days had a high proportion of superficial area covered by transformed tissues after a 7 day-cocultivation duration. These results will help to improve the efficiency of recovery of transgenic plants by increasing the proportion of transformation in the regenerable tissues.

Agrobacterium mediated transfer of chlorsulfuron resistance to commercial flax cultivars.

Agrobacterium tumefaciens carrying a disarmed Ti-plasmid vector containing a chimeric NPT-ll gene and a mutant acetolactate synthase gene (conferring resistance to the herbicide chlorsulfuron) from Arabidopsis was used to transform flax (Linum usitatissimum) hypocotyl tissue. Transgenic regenerants were recovered from the inoculated tissue and were tested for expression of the foreign genes by leaf callus assays on kanamycin and on chlorsulfuron. Transgenic plants were grown to maturity; selfed progeny were similarly tested to determine segregation pattern for the novel genes, and some were grown in chlorsulfuron containing soil. Lines from two major commercial cultivars express chlorsulfuron resistance in greenhouse tests.

Recovery of transgenic plants from "escape" shoots.

The problem of escapes is well known to those investigating the regeneration of transgenic shoots from transformed callus. Shoots can pass various tests and assays for transformation, and are then scored as transgenic, but the progeny do not express the transferred trait and do not contain the T-DNA. Explanations for these enigmatic "escapes" include instability of the T-DNA, genomic rearrangements during meiosis, or merely non-rigorous selection or identification assays giving rise to spuriously positive scorings. At least some shoots, however, are likely to simply be chimeric, containing both transformed and non-transformed cell lines. In this case, the transformed cells are responsible for the positive selection and scoring on tests, but either do not contribute to the germ line (resulting in no transgenic progeny) or contribute to only a portion of the germ line (resulting in many fewer positive segregants than expected). We describe two methods which we used to recover fully transgenic plants from apparent escapes. One method involved analyzing more progeny than would normally be necessary (to identify minority transgenic contribution to the cell line). The other method, (to recover transgenic plants from primary selectants with no transgenic contribution to the germ line) involved regenerating new shoots from leaf tissue used in a selection assay to score the initial shoot as a positive transgenic.

Glyphosate tolerant flax plants from Agrobacterium mediated gene transfer.

Agrobacterium tumefaciens carrying a disarmed Ti-plasmid vector containing a chimeric NPT-II gene and a glyphosate resistance plant-derived 5-enolpyruvylshikimate-3-phosphate synthase gene was used to transform flax hypocotyl tissues. Transformed shoots could be regenerated from the inoculated tissue and were proven to be transgenic by the combination of leaf callus assays, nopaline assays and progeny tests. Co-segregation was observed in the progeny for kanamycin and glyphosate resistance.

Transformed callus does not necessarily regenerate transformed shoots.

Agrobacterium tumefaciens carrying a disarmed Ti plasmid vector was used for the transformation of flax tissue. Transformed callus was obtained from inoculated hypocotyl segments and healthy green shoots were regenerated from this callus. Nopaline assays on shoot tissue were positive for nopaline content if carried out soon after removal of the shoot from the callus but negative if carried out 2-3 weeks after removal. All of these shoots gave rise to kanaymcin sensitive progeny and were most likely escapes arising from non-transformed cells protected from the selective agent by transformed cells in the callus. Careful analysis of regenerated shoots from transformed callus is necessary in order to distinguish escapes from true transgenics.

Salt tolerance through increased vigor in a flax line (STS-II) selected for salt tolerance in vitro.

Progeny of a flax (Linum usitatissimum L. cv "McGregor") plant, regenerated from a cell line selected in vitro for salt tolerance (designated STS-II) was tested for its performance over two generations in normal and in saline soil against its parent variety. Germination, seedling height, flowering, seed set and seed yield in controlled greenhouse conditions were recorded. The putative salt tolerant line was superior in saline soil for all parameters measured, indicating that the mechanism selected in cells in vitro was also active in whole plants, and that the trait is genetically stable and seed transmitted. Unexpectedly, the STS-II line was also superior in the normal, non-stressed soil, indicating that the selected trait is not limited to salt tolerance specifically, suggesting a more general mechanism, such as a general increase in vigor.

A Tissue-Culture Derived Salt-Tolerant Line of Flax (Linum usitatissimum).

Flax seedlings (Linum usitatissimum) were used to initiate callus cultures. After a month of growth, the healthy callus was transferred to a similar medium supplemented with salts to meet or exceed those concentrations found in a saline soil of Saskatchewan. This salt medium also lacked growth hormones. After one month in the salt medium, most of the cells had died, but a few green cell aggregates remained. On non-salt-stress but otherwise similar medium, most cells remained healthy over the same period. The green pockets from the high salt medium were excised and transferred to a different medium that caused shoots to form from the callus. These shoots were later transferred to a medium that caused root formation from the base of the shoots. Plants taken through to maturity in soil have set seed and the progeny of these have been tested for salt-tolerance. Preliminary observations suggest that a salt-tolerant ability is present in the progeny. Some of the advantages and disadvantages of using this method to select for salt-tolerant lines are discussed.

Embryo Formation in Cell Cultures of the Legume Cyamopsis tetragonoloba (Guar).

Seedling tissues of Cyamopsis tetragonoloba (Guar) were cultured on various media to produce calli. These calli sometimes produced organs spontaneously; organogenesis was not controllable on any medium tried. Several calli were transferred to liquid shake cultures to form suspensions. One of these, along with one of the callus cultures, spontaneously turned embryogenic, producing large numbers of somatic embryoids. The embryoids could be removed and germinated on any of several media to plantlet stage, but degeneration of the root primordium precluded further development.

Altered morphogenesis of placental tissues of tobacco in vitro: Stigmatoid and carpelloid outgrowths.

When placental tissue of tobacco (Nicotiana tabacum L.) was cultured on liquid medium with 4% sucrose but with no hormones, many ovules, instead of maturing normally, grew as elongate, style-like structures with terminal stigmas. A limited number of these formed a proximal swelling with ovules. The significance of these results is discussed briefly.