FUNGAL ENDOPHYTES INTRINSICALLY ASSOCIATED WITH MICROPROPAGATED PLANTS REGENERATED FROM NATIVE BOUTELOUA ERIOPODA TORR. AND ATRIPLEX CANESCENS (PURSH) NUTT. JERRY R. BARROW*, PEDRO OSUNA-AVILA, AND ISAAC REYES-VERA USDA, Agricultural Research Service, Jornada Experimental Range, P.O. Box 30003, MSC 3JER, NMSU, Las Cruces, NM 88003-8003 (Received 9 April 2004; accepted 2 August 2004; editor R. J. Newton) Summary Black grama (Bouteloua eriopoda) and fourwing saltbush (Atriplex canescens) are important grass and shrub species in arid rangelands of the northern Chihuahuan Desert. They are naturally colonized by dark septate endophytic fungi that cannot be eliminated by seed disinfestation. Plants were regenerated from both species and appeared to be fungus-free in axenic cultures. Analysis of callus and regenerated plants of both species using dual staining with light and scanning electron microscopy revealed fungal endophytes intrinsically associated with cells, roots and leaves of regenerated plants that are also associated with native plants. Fungal layers and biofilms prevent direct exposure of callus, root or leaf tissues to the external environment. Micropropagation is a valuable tool for identifying key fungal endophytes that enhance drought tolerance in native desert plants. Key words: arid; biofilms; drought; mycorrhizae; symbiosis. Introduction Fungi have been detected in samples of every plant species examined to date (Berbee, 2001). They live within tissues of all plant species and aided in their initial invasion into terrestrial ecosystems (Blackwell, 2000). These relationships vary from pathogens and saprophytes to beneficial mutualists that enhance ecological fitness of plants in native ecosystems (Smith and Read, 1997). Surprising levels of fungal diversity were recently reported in leaves of native trees and in roots of a native grass (Arnold et al., 2000; Vandenkoornhuyse et al., 2002). Asymptomatic fungal endophytes inhabit the apoplastic spaces of above-ground plant parts of cool-season grasses and are vertically transmitted by seed (Clay and Schardl, 2002). Barrow et al. (1997) found that roots of native grasses and shrubs in the warm semiarid rangelands of the southwestern USA were primarily and extensively colonized by dark septate endophytic (DSE) fungi. Barrow and Aaltonen (2001) and Barrow (2003), using dual- staining methodology, analyzed DSE colonization of Atriplex canescens (Pursh) Nutt. (ATCA) and Bouteloua eriopoda Torr. (BOER), important forage shrub and grass species of the northern Chihuahuan Desert. They found that fungal incidence was much greater than previously thought, and that these fungi form a variety of previously unreported atypical and morphologically variable structures. DSE fungi systemically inhabit the entire apoplastic network, form inter- and intracellular structures with cells in both below- and above-ground structures, and are vertically transmitted by seed. Meristem cultures and micropropagated plants have been used to produce plants free of fungi, bacteria, and viruses (Grum et al., 1998; Borkowska, 2002). Unable to produce endophyte-free BOER and ATCA plants by surface disinfestation of seed, we regenerated ATCA plants from meristem cultures and shoot regeneration and BOER plants via somatic embryogenesis. Cells and tissues were examined with scanning electron microscopy (SEM) and were dual stained and analyzed with high-magnification, differential inter- ference microscopy to determine the presence of DSE colonization. Materials and Methods Micropropagation of BOER plants via somatic embryogenesis. Seeds of BOER were harvested from native populations on the USDA-ARS Jornada Experimental Range in the northern Chihuahuan Desert. They were surface- disinfested and germinated on MS (Murashige and Skoog, 1962) medium gelled with agar. Seedling shoots were excised and cultured on MS medium, supplemented with 4.52 mM Dicamba, under light, to induce callus and embryogenic structures. After transferring to MS auxin-free medium, embryo development progressed from globular, torpedo to mature embryos phenotypically identical to those naturally produced in seed (Osuna and Barrow, 2004). Micropropagation of ATCA plants via shoot multiplication. ATCA seeds were surface-disinfested for 1 min in 95% ethanol, 7 min in 2.6% sodium hypochlorite (50% dilution of commercial liquid bleach), and rinsed three times in sterile distilled water. They were placed on hormone-free, modified White’s media for germination (White, 1934). Intact seedlings were transferred to shoot regeneration medium consisting of MS basal salts with 11.42 mM indole-3-acetic acid (IAA) and 18.58 mM 6-furfurylaminopurine (kinetin). The medium was modified using the L2 vitamin formulation of Phillips and Collins (1979) with 30 g l 21 sucrose and solidified with 0.8% agar. The pH was adjusted to 5.8 ^ 0.05 for all media prior to autoclaving at 1218C at 125 kPa for 35 min. Cultures were grown in 100 £ 25 mm *Author to whom correspondence should be addressed: Email jbarrow@ nmsu.edu In Vitro Cell. Dev. Biol.—Plant 40:608–612, November– December 2004 DOI: 10.1079/IVP2004584 q 2004 Society for In Vitro Biology 1054-5476/04 $18.00+0.00 608