PRODUCTION OF ANOMALOUS STRUCTURES IN QUERCUS RUBRA L. CALLUS CULTURES

Internodal stem segments of 2-4-month-old Quercus rubra L. seedlings were cultured on Murashige and Skoog agar medium containing NAA and BA. Structures, termed organoids, were initiated from callus on medium containing 5 mg/l NAA and 0.1 mg/l BA. Organoids consisted of parenchymatous cells with a distinct epidermis and a continuous vascular system. Growth occurred principally by cell division throughout the parenchymatous tissue resulting in a variety of morphological shapes. Although no organized shoot meristems were observed, normal roots were produced; such roots were connected to the vascular system of the organoid. RED OAKS (Quercus rubra L.), major hardwoods of northern midwest forests, valued for their aesthetic and timber qualities, are succumbing to oak wilt induced by the fungus Ceratocystis fagacearum (Bretz) Hunt. Death of infected trees results from the formation of tyloses in the vessels of the xylem (Struckmeyer et al., 1954). Resistance to oak wilt in natural populations of red oak has been observed in Wisconsin (R. D. Durbin, pers. commun.). However, no practical means to increase the number of trees showing resistance to the disease are presently available because seedlings are highly heterozygous and conventional asexual propagation practices are unsuccessful (Skinner, 1952; Flemer, 1962; Jaynes, 1964). In research designed to adapt microculture techniques to the propagation of wilt-resistant red oaks, an apparent anomaly in the development of differentiated shoots from calli was observed. These structures, termed organoids, are described in detail in this report. MATERIALS AND METHODS-Stems were excised from 2-4-month-old, greenhouse-grown red oak seedlings, surface sterilized by dipping in 70% ethanol for 5 seconds, and then transferred to 10% chlorine bleach for 5 min. After three successive rinses in sterile, distilled water, the stems were cut into internodal segments approximately 4 mm in length. The excised tissue 1 Received for publication 9 October 1978; revision accepted 7 May 1979. Research supported by the College of Agricultural and Life Sciences, by the Graduate School, and by the UniversityIndustry Research Program, University of Wisconsin, Madison. The authors express their appreciation to Dr. R. D. Durbin for plant material and invaluable discussions. was cultured on 10 ml of Murashige and Skoog (MS) agar medium (1962) supplemented with 0, 0.01, 0.1, 1, or 5 mg/l benzyladenine (BA) and 0, 0.01, 1, 3, 5, or 10 mg/l a-naphthaleneacetic acid (NAA) in 30-ml glass jars sealed with Parafilm-M. The jars were placed under a 16-hr photoperiod of cool-white fluorescent light at 5 nE! cm2/sec for the 400-700 nm wave band. Tissue for microscopic observation was fixed in the Allen-Bouin formula (Berlyn and Miksche, 1976) for at least 40 hr, dehydrated in a tertiary butyl alcohol series, and infiltrated and embedded in Paraplast. Sections were cut 10 ,um in thickness on a rotary microtome, mounted on glass slides with Haupt's adhesive, and stained with safranin-crystal violet-light green SF (Gerlach, 1969). RESULTS-Stem segments were placed longitudinally on MS agar medium containing all combinations of BA and NAA. After approximately 7 weeks, isolated callus cultures produced anatomically unique structures termed "organoids" (Fig. 1). Organoids formed predominately on calli grown on medium containing 5 mg/l NAA and 0.1 mg/l BA. Although the incidence of organoid initiation was low, no attempt to optimize organoid production was made. Organoids were initiated on callus attached to the original stem segment as well as on subcultured callus. The term "organoids" is used because of the presence of three tissue systems inherent in plant organs, namely, the dermal, the vascular, and the ground tissue (Esau, 1977). However, these organoids differ from true organs in that the former do not appear to originate from organized meristems. Anatomical studies revealed that organoids were initiated from the periphery of calli (Fig. 2).