DEVELOPMENT OF SELF AND HYBRID DATURA EMBRYOS IN ARTIFICIAL CULTURE

Abstract

DATURA INVESTIGATIONS in recent years have included growth of numerous interspecific hybrid embryos in artificial culture. Results obtained with embryos from different species combinations indicated that such embryos were unlike in their response to the nutrient solutions used. The following experiments were une MIcLean, 1946). Seitz-filtered malt extract added to the culture medium was found to promote embryo growth and was substituted for cocoanut milk. MATERIALS AND METHODS.-Seed development of the embryos used in the present investigations has been previously described (Sanders, 1948). They are self embryos of Datura discolor, D. inoxia, D. metel and D. stramonium; hybrid embryos from the compatible cross D. stramonium X D. discolor; and hybrid embryos from five incompatible crosses in which relatively numerous arrested embryos are formed. The incompatible crosses are D. discolor X D. stramonium, D. inoxia X D. discolor, D. inoxia X D. metel, D. inoxia X D. stramonium, and D. metel X D. inoxia. Self embryos of these species grow well in culture when dissected at stages of development ranging from the late heart stage to the mature embryo. Proembryos 0.1 mm. in diameter occasionally grow in culture but as a rule they do not. Embryos for these experiments were, therefore, taken at approximately the early heart stage when there is a real possibility that the embryo will grow and also that it will be more susceptible to changes in the nutrient than at older periods of developmient. At 1 Received for publication June 16, 1949. Contributions from the Department of Botany, Smitlh College, New Series No. 36. The article is taken from a thesis written under the direction of Dr. A. F. Blakeslee and submitted to the faculty of Smith College in partial fulfillment of the requirements for the degree of Doctor of Philosophy. this stage the top of the proembryo has flattened out, cotyledon primordia are just beginning to appear, and the longitudinal axis is about 0.2 mm. Slightly larger embryos were occasionally used because of the difficulty in obtaining the exact stage desired. Degree of embryo development at the time of dissection must be considered since it has been found that growth of embryos in culture varies with the stage of the embryos. Hybrid embryos from the compatible cross (D. stramonium X D. discolor) were also dissected at approximately the early heart stage though in some cases cotyledon differentiation was not evident. Embryos in this cross do not always differentiate normally so that such cases were probably examples either of the lack of normal differentiation or of the delay of differentiation. Hybrid embryos from incompatible crosses, due to the difficulty of obtaining a sufficient number for the experiments, were dissected at the age when the most embryos could be found, old enough for developing embryos to be seen, and young enough for the embryos to be still viable. This age varied with the cross (Sanders, 1948). Embryos were dissected out of the seeds under sterile conditions with the aid of a dissecting microscope and introduced into cotton-plugged vials containing culture media which had been sterilized by autoclaving at 15 lbs. pressure for 20 min. When malt was used in the nutrient, it was sterilized by Seitz-filtering and added to the vials after autoclaving. All nutrients to be compared were used in the same experiment to keep environmental factors affecting growth the same for all. Embryos were introduced into the vials along rows at right angles to the rows representing different nutrients in order to distribute embryos from the same capsule and to balance differences in size and vigor of the embryos among the nutrients. Vials were kept in an incubator at a constant temperature of 26?C. Growth has been expressed as the number of times the embryos increased in size during their days in culture. Size was determined by measuring the longitudinal axis of the embryo or of the largest outgrowth in the cases where multiple growth occurred. Measurements were made the dav of dissection and again after the prescribed number of days in culture. The growth value used is-final average size/original average size. Results are expressed as growth values or as the average of growth values from several experiments. Each experiment was done twice, occasionally three times, with ten embryos per nutrient so that the results are based on at least twenty vials per treatment. Data from individual experiments which were averaged together to obtain the growth values