Temporal competence of embryonic Pinus ponderosa cotyledons to form multiple buds in vitro

To investigate temporal changes occurring during tissue maturation and loss of organogenic competence, embryos of Pinus ponderosa Laws. were excised from seed and placed horizontally on medium in sterile culture. On a hormone-free basal medium, cotyledons differentiated and failed to form multiple buds. When placed on a benzyladenine (BA)-containing medium, the lower cotyledons in contact with the medium formed multiple buds while those elevated above the medium did not. Cotyledons became incompetent to form buds when embryos were initially placed on basal medium for 2 days before being transferred to the BA-supplemented medium. In order to initiate buds, cotyledons of newly excised embryos had to be exposed to BA for at least 3 days. Exposure to BA for longer than 7 days did not significantly increase the number of cotyledonary buds. Cotyledons elevated above the BA-supplemented medium retained organogenic competence to form buds for up to 14 days but did not form buds unless the cotyledons were placed in contact with the medium. The presence of BA is required to retard cotyledon maturation and sustain tissue competence to initiate buds. VEGETATIVE PROPAGATION of many forest conifers is limited by physiological changes occurring during maturation that render mature tissues unable to respond to external organogenic stimuli. Recent progress in tissue culture, however, has permitted the vegetative propagation of several conifers from embryonic or seedling tissues (for review, see David, 1982) and, to a limited extent, bud explants from mature trees (von Arnold and Eriksson, 1979; Bonga, 1982; Chesick, 1985; Gupta and Durzan, 1985). Although juvenile tissue is the most common source of conifer explants, it also undergoes maturation and may only be able to respond to an organogenic stimulus within the first month following germination (Wochok and El-Nil, 1977; Berlyn and Beck, 1980; Skripachenko, 1982; Rumary and Thorpe, 1983). Christianson and Warnick (1985) consider competence as a tissue's ability to respond to an organogenic induction. Conifer cotyledons are therefore competent to respond to the organogenic induction if de novo buds can be induced by exposure to a hormone. Pinusponderosa (ponderosa pine) embryos have been I Received for publication 1 September 1987; revision accepted 26 July 1988. 2 Current address: University of Wisconsin, Department of Horticulture, 1575 Linden Drive, Madison, WI 53706. This research was partially funded by a McIntire-Stennis Research Grant from the School of Forestry of the University of Montana. The authors wish to thank Jeffrey Strachan for help with the statistical analysis, Helena Chambers for assistance in preparing the manuscript, Debbie McNiel for the bud induction illustration, and Barry Flinn and David T. Webb for reviewing the manuscript. chosen to study competence because cotyledons of a single embryo respond differently to the hormone. When an embryo is placed longitudinally on a benzyladenine (BA)-supplemented medium, the shorter lower cotyledons in contact with the medium form de novo buds and meristemoids (nodular meristematic areas), whereas the elevated upper cotyledons elongate and fail to form buds or meristemoids (Ellis and Bilderback, 1984; Fig. 1). Prior to this study, investigations of organogenic competence have used isolated cotyledons (Cheah and Cheng, 1978; Yeung et al., 1981; Patel and Thorpe, 1984; Aitken-Christie et al., 1985; Flinn, 1987). By using intact embryos, the response of genotypically identical cotyledons to an organogenic stimulus can be compared without concern for wound responses generated by excision or environmental differences (regardless of how small) that may be inherent among tissue culture vials. This investigation was designed to determine the minimum and maximum BA exposure times required for bud and meristemoid production by the lower cotyledons of ponderosa pine and to set a temporal boundary for the loss of competence by the lower and upper cotyledons. Defining loss of competence and the onset of determination in a temporal sense is of primary importance if further physiological, biochemical or cytological investigations dealing with the developmental shift from juvenile to mature tissue are to have full impact. MATERIALS AND METHODS-Pinusponderosa (ponderosa pine) seed, obtained from the Mon-