Development of Chloroplast Fine Structure in Aspen Tissue Culture

Chloroplast ontogeny has been examined in 42-day etiolated triploid aspen calltus (Populustremuloides Michx.) subjected to two different light conditions. White and low-intensity red illumination showed little differences in their stimulatory effects on plastid development, the red light-irradiated plastids developing only slightly more slowly. Asynchronous plastid development was noted in both lighting systems. Etioplasts contained an interconnected tubular net, phytoferritin aggregates, electron-transparent vesicles which seem to invaginate from the inner plastid membrane, membrane-bound homogeneous spheroids and starch grains. Irradiation caused various morphological changes within the proplastids; the tubular complex became transformed into the more ordered prolamellar body-like structure from which radiated membrane-bound sacs filled with electron-dense material. These sacs, characterized as thylakoid precursors, were transformed into a thylakoidal system typical of mature chloroplasts. This ontogenetic scheme represents an additional pathway for the development of photosynthetic lamellae. Other lightinduced changes in the developing plastid include disappearance of phytoferritin particles and homogeneous spheroids, decrease in starch content, and appearance of osmiophilic droplets. THE EFFECT of light on the development of chloroplasts is a subject which has been considered in several reviews (Granick, 1961; Goodwin, 1966; Kirk, 1967). The use of tissue culture to study the ontogeny of plastids in higher plants seems worthwhile because it is possible to control the physical and chemical environments and thus regulate differentiation and growth. Recently the ultrastructure of developing chloroplasts in several cultured tissues has been studied (Laetsch and Stetler, 1965; Stetler and Laetsch, 1965; Bergmann and Berger, 1966; Israel and Steward, 1967; Sunderland and Wells, 1968). Our investigation describes the ontogeny of chloroplasts in dark-grown aspen tissue culture stimulated by white and low-intensity red-light irradiation. The sequence of development to be described appears to support the proposal that chloroplast lamellae develop by more than one pathway. MVATERIALS AND METHODS-The triploid aspen callus used in this investigation was initially 1 Received for publication 4 October 1968. Research supported by NSF grants GY-2117 and NSF GB-6066. 2 Present address: Tulane University School of Medicine, 1404 Hawthorne, 1300 Canal Street, New Orleans, Louisiana 70112. isolated from internode stem sections of Populus tremuloides Michx. It has been maintained in tissue culture for 5 years on a basal medium containing major elements, trace elements, 3.0 ,ug/ liter glycine, 0.1 jug/liter thiamine, 0.5,ug/liter NAA, 2 % (w/v) sucrose, and 10 % (v/v) coconut milk (Mathes, 1964). These cultures were incubated in the dark at 27 C. Their only exposure to light during this period was for brief durations, when they were examined for contamination and during aseptic transfer of tissue to fresh petri plates. Tissue explants placed on a no sucrose medium (i.e., same as above except no sucrose added) were dark-grown in petri plates covered with double thickness heavy duty aluminum foil and were incubated at 27 C for 37 days. This tissue was again explanted to a no sucrose medium, and returned to the dark for another 5 days. Callus of uniform texture was selected and once again transferred aseptically to no sucrose plates. A green safelight consisting of a G.E. F15 T8 GG fluorescent tube with Cinemoid plastic sheets #5A, #24, #32, and Mylar-W as filters was used. The filter system provided a transmission spectrum in the range 525-545 m,4. Parallel series were then prepared with these explants as follows: (1) dark-grown explants, (2) explants placed in the