Date of Award
Master of Science
Plant, Soil and Environmental Sciences
Bob. V. Conger
James Caponetti, Effin T. Graham
The objectives of this study were to determine the histological origin of callus or other in vitro tissue initiated from mature orchardgrass embryos, the degree of organization of this tissue, and whether organogenesis and/or embryogenesis occur de novo. The study to determine the tissue of origin was carried out over a 6 day time period. Random samples for histological observation were taken every 24 hr, fixed in a gluteraldehyde solution, and prepared for micro-scopic examination utilizing the paraffin method. The 10 μm sections were stained with safranin 0 followed by the combination stain aniline blue and orange G. The slides were then cover-glassed for microscopic examination. No evidence of callus formation or embryo germination was observed in the 1-day-old cultures. Early germination was apparent in cultures sampled on the second and third day. Four days after plating the embryos, a loose mass of parenchyma cells formed from the basal end of the scutellum near the root cap. The callus samples examined on the fifth and sixth days still showed only a mass of unorganized parenchyma cells. Callus tissue examined at the time of the first subculture (28 days after plating) showed that some of the meristematic centers appeared to arise directly from the embryo, while others appeared to be distinctly separate from the explanted embryo. In some cases the callus appeared to be divided into lobes with meristematic centers located within each lobe. Vascular tissue, primarily xylem initials. were found in the center of the callus lobes and were surrounded by densely stained meristematic centers. At the first subculture the callus was carefully separated from the original explant and transferred to fresh medium. When the callus was approximately 1 cm in diameter, it was divided into three equal size pieces. One piece was fixed in gluteraldehyde for later examina-tion and the other two pieces were subcultured separately in a medium with 1 μM 2,4-D. The original identity of the callus pieces was maintained. When a shoot became visible, the callus piece was removed from culture, fixed histologically, and examined along with the previously fixed sample taken from the same original piece earlier. This allowed comparison of the extent of organization in the callus prior to shoot formation with that after shoot formation. Organized structures such as proembryos in several stages of develop-ment were observed scattered throughout the callus tissue of the earlier sample. Meristematic nodes were also present, but were still in the early stages of development. The later tissue samples with a few shoots on the surface also showed extensive root hair-like growth. Serial sections through this tissue revealed a higher degree of organization than was present in the earlier samples. There were more meristematic centers and nodes present throughout the callus. Many of the meristematic nodes had orderly cell files around them. Strands of vascular tissue were observed in many areas of the callus. New shoot meristems with leaf primordia were visible on the surface of the callus tissue and some were subtended by axillary buds. At the end of the second subculture (84 days), most of the tissue consisted of actively growing meristems and other organized structures. Very little parenchymatous tissue was visible.
McDaniel, Judith Lynn Kerley, "A histological study of the origin, organogenesis, and embryogenesis of callus tissue derived from mature orchardgrass embryos. " Master's Thesis, University of Tennessee, 1981.