Arabidopsis ovule is the target for Agrobacterium in planta vacuum infiltration transformation.

The visual marker GUS has been utilized in this study to understand the Arabidopsis thaliana vacuum infiltration transformation process by Agrobacterium tumefaciens. High transformation frequencies of up to 394 transgenic seeds per infiltrated plant were achieved. The results showed that the majority of the transgenic seeds from single infiltrated plants were from independent transformation events based on Southern analysis, progeny segregation, distribution of transgenic seeds throughout the infiltrated plants and the microscopic analysis of GUS expression in ovules of infiltrated plants. GUS expression in mature pollen and anthers was monitored daily from 0 to 12 days post-infiltration. In addition, all ovules from a single infiltrated plant were examined every other day. GUS expression frequencies of up to 1% of pollen were observed 3-5 days post-infiltration, whereas frequencies of up to 6% were detected with ovules of unopened flowers 5-11 days post-infiltration. Most importantly, transgenic seeds were obtained only from genetic crosses using infiltrated plants as the pollen recipient but not the pollen donor, demonstrating Agrobacterium transformation through the ovule pathway.

An improved green fluorescent protein gene as a vital marker in plants.

A synthetic green fluorescent protein (GFP) gene (pgfp) was constructed to improve GFP expression in plants. Corn and tobacco protoplast transient assays showed that pgfp gave about 20-fold brighter fluorescence than the wild-type gene (gfp). Replacement of the serine at position 65 with a threonine (S65Tpgfp) or a cysteine (S65Cpgfp) yielded 100- to 120-fold brighter fluorescence than wild-type gfp upon excitation with 490-nm light. Incorporation of a plant intron into the coding region yielded an additional 1.4-fold improvement, for a cumulative improvement of about 150-fold in fluorescence at 490-nm excitation. Various versions of pgfp were also stably introduced into corn, wheat, tobacco, and Arabidopsis plants. Bright-green fluorescence was observed with a fluorescence microscope in virtually all examined tissues of transgenic monocots and dicots. In the case of Arabidopsis, expression of the pgfp gene under the enhanced 355 promoter of the cauliflower mosaic virus produced green fluorescence that was readily detectable by eye using a hand-held, long-wave ultraviolet lamp and/or a black-light source.

Stable transformation via particle bombardment in two different soybean regeneration systems.

The Biolistics(®) particle delivery system for the transformation of soybean (Glycine max L. Merr.) was evaluated in two different regeneration systems. The first system was multiple shoot proliferation from shoot tips obtained from immature zygotic embryos of the cultivar Williams 82, and the second was somatic embryogenesis from a long term proliferative suspension culture of the cultivar Fayette. Bombardment of shoot tips with tungsten particles, coated with precipitated DNA containing the gene for ß-glucuronidase (GUS), produced GUS-positive sectors in 30% of the regenerated shoots. However, none of the regenerants which developed into plants continued to produce GUS positive tissue. Bombardment of embryogenic suspension cultures produced GUS positive globular somatic embryos which proliferated into GUS positive somatic embryos and plants. An average of 4 independent transgenic lines were generated per bombarded flask of an embryogenic suspension. Particle bombardment delivered particles into the first two cell layers of either shoot tips or somatic embryos. Histological analysis indicated that shoot organogenesis appeared to involve more than the first two superficial cell layers of a shoot tip, while somatic embryo proliferation occurred from the first cell layer of existing somatic embryos. The different transformation results obtained with these two systems appeared to be directly related to differences in the cell types which were responsible for regeneration and their accessibility to particle penetration.

Agrobacterium-mediated transformation of Solanum tuberosum L. cv. 'Russet Burbank'.

Stem sections from shoot cultures maintained in vitro were used to produce transgenic plants of the potato, Solanum tuberosum L. cv. 'Russet Burbank'. Stem internode pieces inoculated with Agrobacterium tumefaciens containing coat protein genes from potato virus X and potato virus Y, produced shoots with a frequency of 60% in the absence of selection and 10% on medium containing 100 mg/l kanamycin monosulfate. Regenerated shoots were assayed for kanamycin resistance by placing stem segments on callus induction medium containing an increased level of kanamycin. Of a total 255 regenerated shoots, 47 (18%) were kanamycin resistant. Of the kanamycin resistant shoots, 25 (53%) expressed the PVX or PVY coat protein genes as assayed by enzyme-linked immunosorbent assay or Western immunoblot analysis.

Regeneration of soybean (Glycine max L. Merr.) from cultured primary leaf tissue.

A reproducible method for regeneration of plants from primary leaf tissue of 27 varieties of soybean (Glycine max), encompassing maturity groups 00 to VIII, has been developed. Progeny from seeds recovered from regenerated plants appear normal. Best regeneration was from leaf explants (2.1-4.0 mm) obtained from 5 day old seedlings. While 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) was demonstrated to be essential for regeneration, addition of benzyladenine (BA) was found to enhance regeneration. Of the 6 other auxins tested, only picloram induced any regenerative response. Using identical volumes of medium and other conditions, regeneration could be obtained in 95 × 25 mm glass culture tubes but not in 60 × 15 mm Petri dishes.The regeneration of soybeans from primary leaf tissue was shown to be greatly enhanced by pyroglutamic acid (5-oxoproline). Stimulatory effects were attained if pyroglutamic acid was added directly to the medium or if it was formed in situ as a result of chemical transformation of glutamine during autoclaving. The "active" component produced by autoclaving glutamine was not a conjugate of glutamine with inorganic salts or another organic component of the medium. Filter-sterilized glutamine was shown to be inhibitory to regeneration.Murashige and Skoog (MS) and Schenk and Hildebrandt (SH) basal media were compared to Gamborg B5 medium. All contained 0.1 mg/l 2,4,5-T, 40 mg/l adenine sulfate and 10 mM pyroglutamic acid. No regeneration occurred when MS medium was used. Growth and appearance of callus growing on SH and B5 media with the additives were similar. The incidence of regeneration among cultures growing on SH medium was only one third compared to cultures grown on B5 medium.

Plant regeneration by organogenesis in Glycine max.

A procedure for the regeneration of fertile plants by organogenesis from tissue cultures of soybeans, Glycine max is described. Seeds were germinated on reduced inorganic salt MS medium containing 5µM BA. Cotyledonary nodes were excised and cultured on the same medium. Presence of BA in the medium during seed germination and culture of nodal explants was required for multiple shoot and shoot-bud formation. Histological analyses established the de novo nature of shoot regeneration. Separate reduction of the concentration of inorganic salts or substitution of sucrose for fructose during culture had minimal effects on the regeneration response. Conversely, if the BA was reduced, the inhibition response could not be overcome by increased salt concentration or altered carbon source.

Developmental and Biochemical Characteristics of Cold-treated Anthers of Saccharum spontaneum.

The role of cold in stimulating androgenic haploid development was evaluated by assessing morphological and biochemical changes occurring in Saccharum spontaneum anthers during 3 weeks of incubation of panicle sections at 10 °C. During incubation, anthers increased in size; and although many microspores lost viability, those that survived proceeded to divide symmetrically rather than asymmetrically as found in normal microsporogenesis. Anthers which contained dividing microspores differed biochemically from anthers containing non-viable microspores. Anthers initially with high reducing sugars and total free amino acids and with high amylase activity were most likely to produce dividing microspores. Amide metabolism during incubation correlated with microspore development. In all anthers amides increased during cold incubation. The anthers most likely to contain non-viable microspores had much higher asparagine content relative to glutamine content.