Vimla Vasil

Herbicide Resistant Fertile Transgenic Wheat Plants Obtained by Microprojectile Bombardment of Regenerable Embryogenic Callus

We have obtained fertile transgenic wheat plants resistant to the broad spectrum herbicide Basta® (active ingredient phosphinothricin, PPT) by high velocity microprojectile bombardment. The plasmid pBARGUS was used to deliver the selectable bar gene into cells of Type C long–term regenerable embryogenic callus. Phosphinothricin acetyltransferase (PAT) enzyme activity encoded by the bar gene was demonstrated in four independent putative transformed callus lines selected on Basta® from two cultivars. Although somatic embryos and shoots were formed in each of the four lines, plants were recovered only from two. More than 100 green R0 plants were regenerated from the first callus line, of which 40 were grown to maturity. PAT activity was shown in each of the 28 R0 plants tested. Southern analyses confirmed the presence of the bar gene in all of the callus lines, and in each of the R0 and two of the four R1 plants tested. Transformed R0, R1 and R2 plants were resistant to topical applications of Basta®, and the bar gene segregated as a dominant Mendelian trait in R1 and R2 plants.

The Viviparous-1 developmental gene of maize encodes a novel transcriptional activator

The Viviparous-1 (Vp1) gene of maize is specifically required for expression of the maturation program in seed development. We show that Vp1 encodes a 73,335 dalton protein with no detectable homology to known proteins. An acidic transcriptional activation sequence was identified by fusion to the GAL4 DNA-binding domain. Expression of VP1 in maize protoplasts resulted in strong activation (greater than 130-fold) of a reporter gene fused to the promoter of a presumptive target gene. The acidic domain in VP1 was essential for transactivation and could be functionally replaced by the activator sequence of the herpes simplex virus VP16 protein. Our results indicate that VP1 is a novel transcription factor possibly involved in potentiation of a seed-specific hormone response.

Accelerated production of transgenic wheat (Triticum aestivum L.) plants.

We have developed a method for the accelerated production of fertile transgenic wheat (Triticum aestivum L.) that yields rooted plants ready for transfer to soil in 8–9 weeks (56–66 days) after the initiation of cultures. This was made possible by improvements in the procedures used for culture, bombardment, and selection. Cultured immature embryos were given a 4–6 h pre-and 16 h post-bombardment osmotic treatment. The most consistent and satisfactory results were obtained with 30 μg of gold particles/bombardment. No clear correlation was found between the frequencies of transient expression and stable transformation. The highest rates of regeneration and transformation were obtained when callus formation after bombardment was limited to two weeks in the dark, with or without selection, followed by selection during regeneration under light. Selection with bialaphos, and not phosphinothricin, yielded more vigorously growing transformed plantlets. The elongation of dark green plantlets in the presence of 4–5 mg/l bialaphos was found to be reliable for identifying transformed plants. Eighty independent transgenic wheat lines were produced in this study. Under optimum conditions, 32 transformed wheat plants were obtained from 2100 immature embryos in 56–66 days, making it possible to obtain R3 homozygous plants in less than a year.

Improved efficiency of somatic embryogenesis and plant regeneration in tissue cultures of maize (Zea mays L.).

SummaryImmature embryos of eleven cultivars of hybrid maize (Zea mays L.), cultured on 2,4-D-containing nutrient media, showed rapid proliferation of the scutellum and improved efficiency in the formation of embryogenic callus and somatic embryos. High concentrations of sucrose were found to be most favorable for the formation of the embryogenic callus. Embryoids obtained in cultures of all eleven cultivars germinated in vitro and produced normal green plants which were grown to maturity in soil. The effect of genotype and other factors on somatic embryogenesis in maize are discussed.

Transient expression of electroporated DNA in monocotyledonous and dicotyledonous species

Transient expression of electroporated DNA was monitored in protoplasts of several monocot and dicot species by assaying for expression of chimeric chloramphenicol acetyltransferase (CAT) gene constructions. Expression was obtained in the dicot species of Daucus carota, Glycine max and Petunia hybrida and the monocot species of Triticum monococcum, Pennisetum purpureum, Panicum maximum, Saccharum officinarum, and a double cross, trispecific hybrid between Pennisetum purpureum, P. americanum, and P. squamulatum. Recovery and viability of protoplasts after electroporation decreased with increasing voltages and capacitance while CAT activity increased up to a critical combination of voltage and capacitance beyond which the activity dramatically decreased. The optimal compromise between DNA uptake and expression versus cell survival was determined for D. carota and applied successfully to the other species. Maximum transient expression occurred 36 hours after electroporation of D. carota. The potential for using this procedure to rapidly assay gene function in dicot and monocot cells and application of this technique to obtain transformed cereals is discussed.

Overlap of Viviparous1 (VP1) and abscisic acid response elements in the Em promoter: G-box elements are sufficient but not necessary for VP1 transactivation.

The relationship between promoter sequences that mediate Viviparous1 (VP1) transactivation and regulation by abscisic acid (ABA) in the wheat Em promoter was investigated using deletion analysis and directed mutagenesis. The Em1a G-box is strongly coupled to VP1 transactivation as well as to ABA regulation; however, the Em promoter includes additional components that can support VP1 transactivation without ABA responsiveness or synergism. Oligonucleotide tetramers of several G-box sequences, including Em1a, Em1b, and the dyad G-box element from the UV light-regulated parsley chalcone synthase gene, were sufficient to confer VP1 transactivation and the synergistic interaction with ABA to the -45 cauliflower mosaic virus 35S core promoter. These data suggest that VP1 can activate transcription through at least two classes of cis-acting sequences, including the G-box elements and the Sph regulatory motif found in the C1 promoter. The contrasting roles of these motifs in the Em and C1 promoters suggest a basis for the differential regulation of the corresponding genes by VP1.

Plant regeneration from friable embryogenic callus and cell suspension cultures of Zea mays L.

Summary Sectors of a soft, friable, mucilaginous and highly embryogenic callus appeared in embryogenic callus cultures obtained from the scutella of Zea mays L. (maize, commercial hybrid Dekalb XL82) immature embryos. This callus produced globular somatic embryos in very large numbers on the callus maintenance medium and its various modifications. However, the degree of organization in the callus could not be reduced by media alterations, without affecting its embryogenic potential. Stable, rapidly growing and well dispersed cell suspension cultures were obtained from the established friable embryogenic callus. The suspension cultures were comprised of small groups of embryogenic cells, some of which elongated, developed thick walls and ceased to divide. Somatic embryos up to the early scutellar stage were obtained when the suspension cultures were plated on agar-solidified N6 medium containing low concentrations of 2,4-D (0.1−0.2 mg · 1−1). Upon transfer to appropriate agar media somatic embryos in the friable callus, as well as those formed in plated suspension cultures, developed to maturity, and germinated to produce green plants which were successfully transferred to soil.

Isolation and culture of protoplasts of Panicum maximum Jacq. (Guinea grass): somatic embryogenesis and plantlet formation

Summary Protoplasts were isolated from predominantly embryogenic cell suspension cultures derived from proliferating immature embryos and young inflorescences of Panicum maximum Jacq. (Guinea Grass). A large number of protoplasts divided in a liquid culture medium to form hundreds of embryogenic cell aggregates and many globular embryoids. Upon transfer to an agar medium an embryogenic callus was obtained which differentiated more organized embryoids. These embryoids subsequently germinated precociously to form green plantlets. The importance of using embryogenic cell suspensions as a source of totipotent protoplasts of cereals and grasses is emphasized.

Molecular characterization of the fate of transgenes in transformed wheat (Triticum aestivum L.)

Molecular analysis of the transgenes bar and gus was carried out over successive generations in six independent transgenic lines of wheat, until the plants attained homozygosity. Data on expression and integration of the transgenes is presented. Five of the lines were found to be stably transformed, duly transferring the transgenes to the next generation. The copy number of the transgenes varied from one to five in the different lines. One line was unstable, first losing expression of and then eliminating both the transgenes in R3 plants. Although the gus gene was detected in all the lines, GUS expression had been lost in R2 plants of all but one line. Rearrangement of transgene sequences was observed, but it had no effect on gene expression. All the stable lines were found to segregate for transgene activity in a Mendelian fashion.

Identification of callus types for long-term maintenance and regeneration from commercial cultivars of wheat (Triticum aestivum L.)

SummaryImmature embryos, inflorescences, and anthers of eight commercial cultivars of Triticum aestivum (wheat) formed embryogenic callus on a variety of media. Immature embryos (1.0–1.5 mm long) were found to be most suitable for embryogenic callus formation while anthers responded poorly; inflorescences gave intermediate values. Immature embryos of various cultivars showed significant differences in callus formation in response to 11 of the 12 media tested. No significant differences were observed when the embryos were cultred under similar conditions on MS medium with twice the concentration of inorganic salts, supplemented with 2,4-D, casein hydrolysate and glutamine. Furthermore, with inflorescences also no significant differences were observed. Explants on callus formation media formed two types of embryogenic calli: an off-white, compact, and nodular callus and a white compact callus. Upon successive subcultures (approximately 5 months), the nodular embryogenic callus became more prominent and was identified as ‘aged callus’. The aged callus upon further subculture, formed an off-white, soft, and friable embryogenic callus. Both the aged and friable calli maintained their embryogenic capacity over many subculture passages (to date up to 19 months). All embryogenic calli (1 month old) from the different callus-forming media, irrespective of expiant source, formed only green shoots on regeneration media that developed to maturity in the greenhouse. There were no significant differences in the response of calli derived from embryos and inflorescences cultured on the different initiation media. Also, the shoot-forming capacity of the cultivars was not significantly different. Anther-derived calli formed the least shoots. Aged and friable calli on regeneration media also formed green shoots but at lower frequencies. Plants from long-term culture have also been grown to maturity in soil.