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Cotyledons and hypocotyls of Fraser fir [Abies fraseri (Pursh) Poir.] were excised from seeds treated with H 2O 2 for 9 days and placed on bud induction medium containing 10 mg BA/liter and 0.01 mg NAA/liter or medium without growth regulators. Although adventitious buds did not develop, cotyledons exposed to growth regulators responded differently than cotyledons placed on medium lacking growth regulators. Cotyledons and hypocotyls responded similarly to growth regulators during the initial phase in culture, but cell divisions ceased in cotyledons, thus preventing meristemoid and subsequent bud development. After 3 days on medium containing growth regulators cell divisions were localized in epidermal and subjacent layers of hypocotyls, whereas similar cell divisions were’ not observed in hypocotyls placed on medium without growth regulators. Cell clusters consisting of two to five cells (promeristemoids) were present after 7 days on hypocotyls placed on bud induction medium. In hypocotyls placed on medium without growth regulators, stomata continued to develop and cells within the cortex became vacuolated during the first 2 weeks in culture. All explants were transferred to secondary medium after 3 weeks. Cell clusters continued to enlarge into meristemoids on hypocotyls initially placed on bud induction medium. Gradually, meristemoids developed into buds and cataphylls were observed covering bud meristems. Chemical names used: N -(phenylmethyl)-1 H -purine-6-amine (BA), 1-naphthaleneacetic acid (NAA). Adventitious buds can be produced in vitro on hypocotyls of Fraser fir (Saravitz et al., 1987; Saravitz et al., 1991; Zygmont and Schwarz, 1987), but the response is less pronounced compared to other conifers when embryonic tissues are used as explants (Bonga and Durzan, 1987). Also, buds form only on hypocotyls of Fraser fir and very rarely on cotyledons (Saravitz et al., 1991). When placed on medium containing BA and NAA, cotyledons of Fraser fir appear to respond, but buds do not develop (Saravitz et al., 1991). Conversely, adventitious buds produced on hypocotyls elongate into shoots. These shoots can be rooted, but survival of plantlets following transfer to growing medium has not been successful (Saravitz et al., 1991). In vitro adventitious bud differentiation on embryonic explants has been investigated for several conifers, including Douglas fir [ Pseudotsuga menziesii (Mirb.) France] (Kirby and Schalk, 1982), eastern white pine ( Pinus strobus L.) (Flinn et al., 1988), loblolly pine ( Pinus taeda L.) (Mott and Amerson, 1981), Monterey pine ( Pinus radiata D. Don.) (Thorpe, 1988), Norway spruce [ Picea abies (L.) Karst.] (von Arnold and Hakman, 1988), and black ( Picea mariana Mill.) and white [ Picea glauca (Moench) Voss] spruce (Rumary et al., 1986). Generally, these studies have shown that cell divisions leading to adventitious bud development are stimulated by various growth regulators, most notably cytokinins, and the divisions are restricted to epidermal and subjacent cells of the explants. Initial cell divisions for publication 14 Jan. 1992. Accepted for publication 29 July 1992. rch was funded by the North Carolina Agr. Res. Serv. (NCARS), C 27695-7643. Use of trade names in this publication does not imply nt by the NCARS of products named nor criticism of similar ones ned. Assistance of the North Carolina Forest Service is gratefully ged. From a thesis submitted by C.H.S in partial fulfillment of the ts for the PhD degree. The cost of publishing this paper was defrayed the payment of page charges. Under postal regulations, this paper ust be hereby marked advertisement solely to indicate this fact. raduate Research Assistant. Currently, Postdoctoral Research Assot. of Soil Science. Professor, Dept. of Forestry. . Soc. Hort. Sci. 118(1):163-167. 1993. occur resulting in cell clusters termed promeristemoids (Flinn et al., 1988; Villalobos et al., 1985). Further development of promeristemoids results in meristemoids that gradually give rise to buds. It is intriguing to speculate whether similar histologicalchanges occur in hypocotyls of Fraser fir cultured in vitro. In addition, anatomical research with Fraser fir might explain why adventitious bud formation on hypocotyls is generally low and virtually nonexistent on cotyledons. Therefore, we examined the histological changes that occur when excised cotyledons and hypocotyls of Fraser fir are placed on bud induction medium. Materials and Methods Explant source, seed germination, and culture conditions. Seeds were collected in 1981 by the North Carolina Forest Service from native trees at Roan Mountain (36°01' N latitude, 82°05' W longitude, elevation = 1900 m), dried to a moisture content of 4% to 6%, and stored in a polyethylene bag at -17C. Following removal from storage, seeds were immersed in 70% ethanol for 30 sec to remove surface resin, nicked at the micropylar end with a scalpel, and initially placed in 1% (v/v) H 2O2 for 3 days. On the 4th day, seeds were transferred to 0.03% (v/ v) H2O 2 and the concentration remained constant thereafter. Regardless of concentration, H2O2 solutions were changed each day. As a further measure to prevent contamination, seeds were treated daily, beginning on day 4, with either 6% (v/v) H202 for 5 min or 70% (v/v) ethanol for 30 sec. These procedures were alternated daily and were used in addition to the H2O 2 treatment (Saravitz et al., 1991). During H2O2 treatment, seeds were maintained at an 8/16 h thermoperiod of 30/20C and were subjected daily during the 8-h cycle to irradiation from a combination of cool-white fluorescent lamps and incandescent bulbs. The lamps and bulbs provided a photosynthetic photon flux [PPF (400 to 700 nm)] of 17.9 μmol·m ·s -1 (1.3 klx). This and all other light measurements were recorded with a LI-COR LI 185A quantum/radiometer/photometer (LI-COR, Lincoln, Neb.). Following 9 days of treatment with H2O 2, seedlings were

Histology of In Vitro Adventitious Bud Development on Cotyledons and Hypocotyls of Fraser Fir