Plant Cell Reports (1996) 15:829-832 Plant Cell Reports Springer-Verlag 1996 Androgenesis of the salt tolerant shrub Atriplex glauca L. Kenny ~ and P. D. S. Caligari 2 Institut Agronomique et V~t6rinaire Hassan II, BP 438/18s Complexe Horticole, Agadir, Morocco 2 Department of Agricultural Botany, School of Plant Sciences, The University of Reading, PO Box 221, Reading, RG6 6AS, UK Received 20 April 1995/Revised version received 12 December 1995 - Communicated by M. R. Davey ABSTRACT Clusters of flowers, 4 to 5 days old, were successfully cultured on Nitsch and Nitsch (1969) medium in the presence of growth regulators. An average of 20 plants per explant were produced after 6 weeks. Organized growth was obtained only from flowers collected during spring or early summer. Microscopic observations during shoot regeneration indicated that shoot organogenesis took place directly from microspores to give rise to shrubby plants. Their subsequent transfer onto fresh medium in the presence of a different set of growth regulators resulted in the production of "haploid" and "diploid" lines in the proportion of 50:50. The presumed haploid plants were successfully rooted on Woody Plant Medium (Lloyd and McCown 1980) within a week. The significance of androgenic lines for the study of salt tolerance and the genetic improvement of Atriplex forage quality is discussed. ABBREVIATIONS 2,4-D - Dichlorophenoxyacetic add; 2iP- 2-Isopentenyladenine; BA - Benzylaminopurine; IAA - Indoleacetic acid; KIN - Kinetin; MS - Murashige and Skoog (1962); NAA - Naphthaleneacetic acid; NN - Nitsch and Nitsch (1969); WP - Woody Plant Medium (Lloyd and McCown 1980). INTRODUCTION Plants of the genus Atriplex have been used as models in several physiological studies of salt tolerance (Ungar 1991). They are also of interest for forage and fuel production particularly in arid salt- affected areas (Choukrallah 1991). Considerable progress has been made in domesticating several Atriplex species for this purpose (Le Houerou 1992). Tissue culture technology has been recently applied for mass cloning of elite genotypes ofA. canescens (Wurtele et al. 1987) and A. halimus (Kenny 1993). The present work describes an androgenic system developed for A. glauca, and its significance for micropropagation and as a breeding tool is discussed. MATERIALS AND METHODS Single flowers, 4 to 5 days old, or a cluster of 4-5 flowers were collected from three year old shrubs of A. glauca grown at the experimental farm of the "Complexe Horticole" in Agadir, Morocco. The explants were surface sterilised with 5% sodium Correspondence to: P. D. S. Caligari hypochlorite and rinsed three times with distilled, autoclaved water before use. Five explants were placed in each Magenta TM GA7-3 vessel containing 50 ml of nutrient medium. The pH of all nutrient media was adjusted to 5.7 5: 0.1, prior to autoclaving, using HCI (1 N) or KOH (1 N) and solidified with Dlfco bacto agar (7%). Cultures were kept at a temperature of 28~ and a light intensity of 90 to 100 #tool m -2 s-1 for 16 h day "1. Experiment 1. Flowers were first collected during the autumn of 1992 and grown on two nutrient media: Murashige and Skoog (1962) and Nitsch and Nitsch (1969), both supplemented with glucose (30 g 11) and 2iP at 0, 0.01 and 0.1 mg Nin combination with 2,4-D at the same concentrations. Each treatment was replicated 10 times in a completely randomised design. A similar experiment was repeated in the spring of 1992 as flowers collected during the autumn gave no organised morphogenetic response. Vitrified (= hyperhydric according to the new definition suggested by Debergll et al. 1992) shoots were transferred onto half and full strength MS medium containing 400 mg 1 -~ of NaC1 and low concentrations of sucrose at 2.5 and 10 g 1-1in an attempt to solve the problem of hyperhydricity (vitrification). Experiment 2. The effect of 2,4-D, NAA and IAA at 0.01 mg 1 -~ was re-evaluated on NN medium supplemented with ZiP at 0, 0.01 and 0.1 mg 11. Fresh flowers were collected in the summer of 1992 from the same genotype and prepared as indicated previously. After 6 weeks shoots were transferred for a second subculture onto a fresh NN medium containing the same glucose and agar concentration mentioned above and supplemented with IAA at 0.1 mg 1-1 associated with 0.1 mg 1-1 of KIN and 2iP. Normal (non-hyperhydric) plantlets were produced after four weeks and transferred to WP for rooting. Sections of flower cultures were sampled, four per treatment, after 10 weeks, mounted on grids and stained with toluidine blue (1% w/v in 1% borax solution) for microscopic examination. RESULTS Flowers collected during the autumn gave no in vitro responses on MS and NN media irrespective of the hormone concentrations used. All explants died by the end of the second week. Similarly, single flowers harvested either in autumn, spring or summer gave no response in any the media x hormone combinations tested. Only when exp/ants were collected in