GROWTH AND MORPHOGENESIS OF GLOBULAR AND OLDER EMBRYOS OF CAPSELLA IN CULTURE

RAGHAVAN, V. , and J. G. TORREY. (Harvard U., Cambridge, Mass.) Growth and morphogenesis of globular and older embryos of Capsella in culture. Amer. Jour. Bot. 50(6): 540-551. Illus. 1963. -Early heart-shaped and older embryos of Capsella bursa-pastoris (>80,u long), when cultured aseptically in a semi-solid medium containing mineral salts, vitamins, and 2% sucrose, developed into small plantlets. When grown in a 12-hr light/12-hr dark cycle (light-grown cultures), formation of the primary root system was suppressed in embryos 1000l long, the inhibition of formation of the root system in light did not occur. Growth of the shoot apex of intermediate and torpedo-shaped embryos was slightly accelerated in light. There was a correlation between the rate of growth of the embryos and their initial ength. Better growth in length was observed in light-grown than in dark-grown cultures. Supplementing the basal medium with indoleacetic acid, kinetin, and adenine sulfate permitted successful development of globular embryos (<80M long) in vitro. Although a high sucrose concentration or a high salt concentration i the medium partially replaced the effect of the growth factors in inducing development of the globular embryos, no absolute requirement for a high osmotic concentration in the medium was found. The results favor caution in interpreting the inability of small embryos to grow in terms of the osmotic conditions of the culture milieu. THE FACTORS that control the progressive and orderly development of embryos throughout heir ontogeny have attracted the attention of experimental embryologists for some time (Needham, 1942; Brachet, 1950; Wardlaw, 1955). There is now substantial indirect evidence that growth and differentiation of plant embryos may result from an induction by gradients of nutritional substances and hormonal factors in the immediate environment of the developing embryos (Rijven, 1952; Wardlaw, 1955). In the early stages of embryogeny, embryos develop at the expense of the surrounding endosperm tissue, and as far as the evidence goes, such embryos are highly heterotrophic in character. Attempts to culture very small plant embryos, especially at the morphological stages designated as "globular" and "heartshaped," in chemically defined nutrient media outside the plant tissues have met with little success (Van Overbeek, Conklin and Blakeslee, 1942; Rijven, 1952; Norstog, 1961; Swamy, 1961). It is only after the embryos have developed within the ovular tissues to the beginning of cotyledonary development and the attainment of bilateral symmetry that they become sufficiently independent and autotrophic to lend themselves to culture in vitro even in complex nutrient media. While the nutritional requirements of young embryos are complex, and their responses often highly variable, progressively older embryos show less rigorous requirements and more definitive responses to added nutrients in the medium. 1 Received for publication November 8, 1962. 2 This work was supported by a grant (G-18684) from the National Science Foundation. The genus Capsella has been a favorite object for both descriptive and experimental embryology since the work of Hanstein (1870). Later, Famintzin (1879), Schaffner (1906), and Soueges (1914, 1919) made more thorough studies of the embryogenesis of C. bursa-pastoris. The first published report of in vitro culture of the embryos of this species was by Rijven (1952). A modification of Rijven's classification and terminology concerning embryo stages will be used in this paper (Table 1). Rijven found that growth in culture of the embryos excised from the ovules during the course of development depended upon the presence in the medium of amino acids which apparently regulated the metabolic processes involved in growth. In addition, Rijven (1952, 1955, 1956) observed that the addition of glutamine produced a stimulation of growth which was even greater than that obtained in the presence of casein hydrolysate or a complex mixture of amino acids of the composition of edestin. Nevertheless, Rijven did not succeed in cultivating embryos less than 100lu in mean initial length. Embryos at the heart-shaped or torpedo-shaped stages (140700,u long) responded consistently to the addition of nutrients only when grown in hanging-drop culture in a medium of high osmotic pressure obtained by the addition of 12-18% sucrose. More recently, using Rijven's experimental procedure, Veen (1961, 1962) studied the effects of substances like gibberellic acid and kinetin on the growth of Capsella embryos in culture. Embryos of this species have also been used for cyto-histochemical analysis of embryogenesis (Pollock and Jensen, 1961, 1962).