Genetic Markers Associated with Green and Albino Plant Regeneration from Embryogenic Barley Callus

the resultant improvement in regenerability, have been instrumental in the transformation of once-recalcitrant Genetic control of plant regeneration from cultured plant tissues barley genotypes, and in increasing the efficiencies of has been documented for a number of species. The characterization and manipulation of loci that influence morphogenic responses may transformation of amenable genotypes (Lemaux et al, be useful for the development of highly regenerable germplasm or 1999). for physiological investigations. Quantitative trait loci (QTLs) for A genetic approach to improving plant regeneration morphogenesis from barley cell cultures were identified on the basis would be selection for the accumulation of favorable of associations of mapped markers with the regeneration responses alleles for regenerability into a single, presumably supeof 77 doubled haploid (DH) lines derived from the cross Steptoe/ rior, genotype. Plant regeneration from cultured tissues Morex. Two models were developed, one describing green plant rehas been shown to be under genetic control in a number generation, and one describing albino plant regeneration, measured of species, and genetic markers associated with plant as the numbers of green and albino plants regenerated per gram fresh regeneration have been identified in cereals such as weight of embryogenic callus. Approximately 62 and 12% of the barley (Komatsuda et al., 1995; Mano et al., 1996), observed variability for green and albino plant regeneration, respectively, was explained by the models. An independent data set was wheat, (Triticum aestivum L.; Ben Amer et al., 1997), developed that consisted of the regeneration responses of 25 addirice, (Oryza sativa L.; He et al., 1998), and maize (Zea tional DH lines which were chosen randomly from the same segregatmays L.; Armstrong et al., 1992). Several examples of ing population. The models were tested for their ability to predict genetic manipulations of regenerability exist. Regenthe responses of these independent DH lines. This study identified erability characteristics have been transferred via hynew QTLs for plant regeneration (one for green plants and at least bridization and selection in maize (Armstrong et al. one for albino plants), and confirmed previously reported associations 1992) and in barley (Komatsuda et al., 1995). In the of three QTLs with green plant regeneration. study by Komatsuda et al. (1995), a single locus was identified; however, a subsequent study identified three additional loci influencing plant regeneration from barC techniques for the production of genetiley callus (Mano et al., 1996). Further work to identify cally transformed barley (Hordeum vulgare L.) and study the loci linked to these QTLs may lead to plants are heavily dependent on the delivery of DNA a greater understanding of the physiological processes into totipotent cells within cultured tissues. Such cells involved in the growth and differentiation of somatic must be induced to differentiate into fertile plants foltissues. Syntenic relationships among cereal species lowing a mutagenic period of in vitro growth and selec(Van Deynze et al., 1995) suggest that the results of tion. Transgenic cells of most barley genotypes fresuch studies will be broadly applicable to many species. quently cannot be induced to differentiate into plants, The objectives of this study were to identify QTLs or only albino plants can be recovered (for a recent affecting green and albino plant regeneration from barreview, see Lemaux et al., 1999). Ultimately, it will be ley callus. We identified at least two previously unredesirable to introduce exogenous DNA without relying ported QTLs for morphogenesis in cultured barley tison tissues which are genetically and epigenetically unsues, and provide confirmation of three of the four stable (for example, see Phillips et al., 1994; Bregitzer previously reported QTLs (Komatsuda et al., 1995; et al., 1998b); in the meantime, greater transformation Mano et al., 1996). In addition, predictive models were efficiencies have been realized by improving existing developed and tested for use in marker-facilitated selectissue culture and transformation protocols. tion schemes. One successful approach to barley transformation has been simply to utilize particular genotypes that are ameMATERIALS AND METHODS nable to the requirements of transformation, such as Explant Donor Growth Conditions Golden Promise (Wan and Lemaux, 1994) or Igri (Jähne et al., 1994). Another approach has been to modify the Seventy-seven DH lines and their parents were chosen ranprotocols used for culturing barley tissues; for instance, domly from the mapping population developed from the cross optimizing copper concentrations and autoclaving proof Steptoe/Morex (Kleinhofs et al., 1993), and grown in growth cedures have each been shown to increase the recovery chambers as previously described (Bregitzer et al., 1995). Three separate evaluations, separated by time, were conof green plants by a factor of two (Dahleen, 1995; Bregducted for each DH line. Because a single growth chamber itzer et al., 1998a). Increased copper levels combined could not accommodate all 77 DH lines, they were assigned with particular combinations of phytohormones, and at random to one of three growth chambers. Each of the three growth chambers included the Steptoe and Morex parents USDA-ARS, P.O. Box 307, Aberdeen, ID 83210. Received 25 Jan. 2000. *Corresponding author ([email protected]). Abbreviations: QTLs, quantitative trait loci; DH, doubled haploid; gfw, gram fresh weight. Published in Crop Sci. 41:173–179 (2001).