Indra K. 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.

Plant cell and tissue culture.

Part A: 1. Initiation, Nutrition and Maintenance of Plant Cell and Tissue Cultures F. Constabel. 2. Morphogenesis and Regeneration T.A. Thorpe. 3. Meristem and Shoot Tip Culture: Requirements and Applications N.S. Nehra, K.K. Kartha. 4. Plant Protoplasts for Cell Fusion and Direct DNA Uptake: Culture and Regeneration Systems A. Fehrer, D. Dudits. 5. Isolation and Characterisation of Mutant Cell Lines P.J. Dix. 6. Origins, Causes and Uses of Variation in Plant Tissue Cultures A. Karp. 7. Production and Use of Isogenic Lines G. Wenzel, B. Foroughi-Wehr. 8. In vitro Methods for the Control of Fertilization and Embryo Development V. Raghavan. 9. Cryopreservation and Germplasm Storage K.K. Kartha, F. Engelmann. 10. Plant Transformation M.A.W. Hinchee, D.R. Corbin, C.L. Armstrong, J.E. Fry, S.S. Sato, D.L. Deboer, W.A. Petersen, T.A. Armstrong, D.V. Connor-Ward, J.G. Layton, R.B. Horsch. 11. Cell Culture for Production of Secondary Metabolites F. Constabel, R.T. Tyler. Part B: 12. In vitro Culture of Cereals and Grasses I.K. Vasil, V. Vasil. 13. In vitro Culture of Legumes M.R. Davey, V. Kumar, N. Hammatt. 14. In vitro Culture of Vegetable Crops J.F. Reynolds. 15. In vitro Culture of Potato M.G.K. Jones. 16. In vitro Culture of Root and Tuber Crops A.D. Krikorian. 17. In vitro Culture of Oilseeds C.E. Palmer, W.A. Keller. 18. In vitro Culture of Temperate Fruits R.H. Zimmerman, H.J. Swartz. 19. In vitro Culture of Tropical Fruits J.W. Groisser. 20. In vitro Culture of Plantation Crops A.D. Krikorian. 21. In vitro Culture of Forest Trees I.S. Harry, T.A. Thorpe. 22. In vitro Culture of Ornamentals P. Debergh. Index.

Developing cell and tissue culture systems for the improvement of cereal and grass crops

Summary Recent advances in cell culture and molecular biology of higher plants, which are key components of plant biotechnology, have demonstrated the considerable power and potential of these technologies in the genetic modification and improvement of plants that can not be accomplished by conventional genetic methods. This has stimulated a great deal of interest and activity in university as well as corporate research laboratories. Nevertheless, the fact remains that most of the success achieved so far has been with model plant species and the transfer of these new technologies to major crop species that are the principal targets of biotechnology has either been slow and difficult, or is non-existent. In order to have any meaningful impact on agriculture the developing biotechnology must be equally and readily applicable to important crop species. The cereals and grasses, which constitute the most important group of crop plants, have until recently been found to be very recalcitrant to cell culture techniques. This article describes the advances made in cell culture of important members of this group of crop species. It highlights the success achieved in establishing totipotent callus and cell suspension cultures, and reports the development of protoplast culture systems yielding somatic embryos and plants and the recent recovery of somatic hybrid cell lines and genetically transformed cell lines. The importance of the age and physiological state of the explant, and the relative genetic stability of embryogenic cultures and regenerated plants is discussed.

Plant tissue culture media

Plant tissue culture techniques have become vitally important for pursuing a wide range of fundamental and applied problems in research and development. The techniques encompass a variety of procedures used for specific purposes. The growing of masses of unorganized cells (callus) on agar or in liquid suspension is widely employed in biochemical and growth studies (1-5). The culture of segments of stems, roots, leaves or of callus provides systems to study differentiation, morphogenesis and plant regeneration (6, 7). Shoot apex culture methods leading to plant regeneration have been adopted for plant propagation and production of virusfree stock (8). The culture of anthers and pollen provides new approaches to haploid plant formation (9). Recently the technology has been extended to include the isolation and culture of plant protoplasts which are employed in fusion and somatic cell hybridization (10-13). The development of the various types of tissue culture has been based on empirical approaches, and some of the observations recorded in the literature may not be typical for plant cells. Differences in medium, environment, age, cell origin, and growth rates may explain the behavior of a particular line and need not represent a general characteristic of plant cells in culture. More uniformity in conditions of culture would assist in making data and observations more comparable.

Plant regeneration from cultured immature embryos and inflorescences ofTriticum aestivum L. (wheat): Evidence for somatic embryogenesis

SummaryTissue cultures ofTriticum aestivum L. (wheat) initiated from young inflorescences and immature embryos possessed the potential for regeneration of whole plants. Both a friable and a compact type of callus were produced on Murashige and Skoogs medium with 2 mg/l 2,4-dichlorophenoxyacetic acid. The friable callus contained meristematic centers in which the peripheral cells ceased dividing, elongated, and could be easily separated. Roots were frequently formed in this type of callus. The compact, yellowish, and nodular callus arose from the epithelial and sub-epithelial cells of the embryo scutellum, and the rachis and glumes of the young inflorescence. Such callus had a smooth surface and characteristic chlorophyllous areas. Plants were regenerated only from the compact callus. The first sign of differentiation in the compact callus was the formation of a cleft or notch on the smooth surface, followed by the appearance of trichomes and the direct development of leafy structures which were not associated initially with any shoot meristems. Multiple shoots subsequently arose at the bases of the leafy structures, which are considered modifications of the scutellum, a definitive part of the cereal embryo. Accordingly, we suggest that while typical bipolar embryos are generally not formed, plant regeneration nevertheless takes place through embryogenesis and the precocious germination of the embryoids. Plants regenerated from immature embryo and inflorescence cultures were grown to maturity in soil, and were shown to have the normal chromosome number of 2n=6x=42.

DNA-based markers in plants

General preface. Preface. 1. Some concepts and new methods for molecular mapping in plants* B. Burr. 2. PCR-based marker systems R. Reiter. 3. Constructing a plant genetic linkage map with DNA markers N.D. Young. 4. Use of DNA markers in introgression and isolation of genes associated with durable resistance to rice blast D.-H. Chen, et al. 5. Mapping quantitative trait loci S.J. Knapp. 6. Comparative mapping of plant chromosomes A.H. Paterson, J.L. Bennetzen. 7. Breeding multigenic traits C.W. Stuber. 8. Information systems approaches to support discovery in agricultural genomics B.W.S. Sobral, et al. 9. Introduction: molecular marker maps of major crop species R.L. Phillips, I.K. Vasil. 10. Molecular marker analyses in alfalfa and related species E.C. Brummer, et al. 11. An integrated RFLP map of Arabidopsis thaliana* H.M. Goodman, et al. 12. An integrated map of the barley genome A. Kleinhofs, A. Graner. 13. DNA-based marker maps of Brassica, C.F. Quiros. 14. Molecular genetic map of cotton A.H. Paterson. 15. Maize molecular maps: Markers, bins, and database E.H. Coe, et al. 16. RFLP map of peanut H.T. Stalker, et al. 17. Phaseolus vulgaris - The common bean integration of RFLP and RAPD-based linkage maps C.E. Vallejos, et al. 18. RFLP map of the potato C. Gebhardt, et al. 19. Rice molecular map S.R. McCouch. 20. A framework genetic map of sorghum containing RFLP, SSR and morphological markers J.L. Bennetzen, et al. 21. RFLP map of soybean R.C. Shoemaker, et al. 22. Genetic mapping in sunflowers S.J. Knapp, et al. 23. The molecular map of tomato A. Frary, S.D. Tanksley. 24. Molecular-marker maps of the cultivated wheats and other Triticum species G.E. Hart. 25. Molecular marker linkage maps in diploid and hexaploid oat (Avena sp.) S.F. Kianian, et al. 26. A compilation of molecular genetic maps of cultivated plants O. Riera-Lizarazu, et al. List of Contributors. Subject Index.

Isolation and culture of cereal protoplasts : Part 2: Embryogenesis and plantlet formation from protoplasts of Pennisetum americanum.

SummaryProtoplasts isolated from embryogenic suspension cultures derived from immature embryos of pearl millet (Pennisetum americanum) gave rise to cell masses. These cell masses upon transfer to a hormone-free medium formed embryoids, which further developed into plantlets with roots and shoots.

Somatic embryogenesis in sugarcane (Saccharum officinarum L.). I: The morphology and physiology of callus formation and the ontogeny of somatic embryos

SummaryEmbryogenic callus was induced on segments of young leaves of sugarcane (Saccharum officinarum L.) cultured on Murashige and Skoogs medium supplemented with 0.5–3.0 mg/2,4-D, 5% coconut milk and 3–8% sucrose. The fourth and fifth leaves, especially their midrib and sheath regions within 5 cm from the leaf base, were most suitable for the induction of embryogenic callus. Many embryoids (= somatic embryos) were formed when the callus was transferred to low 2,4-D media (0.25–0.5 mg/l), or was allowed to remain on the high 2,4-D medium for a prolonged period. Plantlets obtained from the germination of embryoids were transferred to soil and grown to maturity.The embryogenic callus was formed by divisions in mesophyll cells situated primarily in the abaxial half of the leaf, and also from cells of the vascular parenchyma. The embryoids developed by internal segmenting divisions in single richly cytoplasmic cells located at the periphery of the embryogenic callus and showed the typical organization of grass embryos.

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.