Development of Transformation Systems for Elite Barley Cultivars

Phil Bregitzer¹, Robert D. Campbell¹, Lynn S. Dahleen², Peggy G. Lemaux³,

and Myeong-Je Cho³

¹USDA-ARS, Aberdeen, Idaho, USA; ²USDA-ARS, Fargo, North Dakota, USA

³Dept. of Plant and Microbial Biology, University of California, Berkeley, CA, USA

Non-sexual transfer of DNA to cereal species such as barley (Hordeum vulgare) has historically been problematic, chiefly because of problems encountered during attempts to regenerate cultured transgenic cells. Frequently, few or no plants can be recovered following selection for transformed cells, or only albino plants can be recovered (for a review, see Lemaux et al., 1999), and the transformation process exacerbates the mutational process(es) that lead to somaclonal variation (Bregitzer et al., 1998a; Choi et al., 2000). Because problems with regeneration have been one of the major obstacles to obtaining transgenic barley plants, researchers have focused on the transformation of particular cultivars that are especially suited to in vitro conditions. Great success has been had with cultivars such as Golden Promise and Igri that can regenerate green, fertile plants from transformed cells with relatively high efficiency.

When the stated purpose of a transformation project is to produce germplasm with relevance to particular commercial applications, however, there is more to consider than the inherent regenerability of transgenic cells. Introgression of transgenes into commercially relevant germplasm will be faster and easier if the transgenic parent and commercial germplasm are genetically similar. Certain transgenic modifications will be greatly influenced by the genetic background in which they are expressed. For instance, QTLs for Fusarium head blight (FHB) resistance have been shown to cosegregate with certain morphological characteristics (Zhu et al., 1999). Thus, transgenically-mediated genetic modifications to obtain FHB resistance cannot be optimally evaluated in a two-rowed Golden Promise background when the goal is to produce FHB-resistant 6-rowed cultivars.

The elite, 6-rowed, U.S. cultivar Morex was one cultivar chosen for study. It represents a significant pool of commercial germplasm, and had in our experience extremely poor regeneration characteristics. Initial experiments involved optimization of 2,4-D levels (Bregitzer et al., 1995). Dahleen (1995) discovered that surprisingly high levels of copper included in the culture medium facilitated regeneration of green plants; for Morex, increasing copper from 0.1 (standard MS level) to 5 µM increased plant regeneration approximately 2.1-fold (Bregitzer et al., 1998b). The same study also reported a 1.7-fold increase in regenerability as a result of avoiding undesirable chemical reactions by separating certain medium components during autoclaving. Another step forward in our ability to work with Morex came with the discovery of heterogeneity for regenerability within this cultivar, and a single plant-derived line (Morex2) was selected based on its relatively high regenerability. Data derived from two separate experiments are shown in Table 1. This selection does not have any apparent, significant difference from Morex in agronomic performance (Table 2). Cho et al. (1998) discovered that appropriate use of 6-benzyl adenine had a significant and positive effect on regeneration from barley, and was able to transform the elite cultivars Galena and Harrington. Finally, two of us (Dahleen and Bregitzer, manuscript in preparation) have been making adjustments to individual micronutrients in the standard MS medium and in some cases (iron and boron) we have seen significant improvement in regeneration.

The media regime adopted by Dahleen and Bregitzer incorporates significant changes for initation, maintenance, and regeneration. In brief, the original protocol was to culture embryos on initiation medium (4 weeks), subculture callus to maintenance medium (biweekly subcultures), and regenerate plants with a two-step process (2 weeks on step 1 and 4 weeks on step 2). The improved protocol calls for 4 weeks on initiation medium, subcultures at 3 weeks on maintenance medium, and 4 weeks each on regeneration steps 1 and 2. Variations in the timing of callus transfers from one medium to another can be made depending on individual preferences; modifications to the media formulations are of more importance (Table 3). In tests done on the cultivars Baronesse, Colter, Conlon, Crystal, Foster, Golden Promise, Harrington, and Morex, and the breeding lines ND15477 and 90Ab321, the new medium increased the regenerabilty at least one order of magnitude, and in some cases two orders of magnitude; for instance, Golden Promise regenerated 10.5 (old) and 101.7 (new) green plants per petri dish, and Morex2 0.25 (old) and 38.9 (new) green plants per petri dish. Attempts to transform Morex and Conlin have met with limited success (Bregitzer, Dahleen, unpublished), and characterizations of existing transgenic plants are in progress.

^aJulian date

^bdata only from Tetonia, 1997

^cdoes not include data from Casselton, Langdon, or Prosper in 1996

^ddoes not include data from Casselton, Langdon, Prosper, or Minot in 1997.

^edata from Casselton, Langdon and Prosper, ND (1996 and 1997), Minot, ND and Tetonia, ID (1997)

^aas modified by Bregitzer, 1992

References

Bregitzer, P. 1992. Plant regeneration and callus type in barley: effects of genotype and culture media. Crop Sci. 32:11-8-1112.

Bregitzer, P., R.D. Campbell, and Y. Wu. 1995. Plant regeneration from barley callus: Effects of 2,4-dichlorophenoxyacetic acid and phenylacetic acid. Plant Cell Tiss. Org. Cult. 43:229-235.

Bregitzer, P., S.E. Halbert, and P.G. Lemaux. 1998a. Somaclonal variation in the progeny of transgenic barley. Theor. Appl. Genet. 96:421-425.

Bregitzer, P., L.S. Dahleen, and R.D. Campbell. 1998. Enhancement of plant regeneration from embryogenic callus of commercial barley cultivars. Plant Cell Rep. 17:941-945.

Cho, M.-J., W. Jiang, and P.G. Lemaux. 1998. Transformation of recalcitrant barley cultivars through improvement of regenerability anddecreased albinism. Plant Sci. 138: 229-244.

Choi, H.W., P.G. Lemaux, and M.-J. Cho. 2000. Increased chromosomal variation in transgenic versus nontransgenic barley (Hordeum vulgare L.) plants. Crop Sci. 40:524-533.

Dahleen, L.S. 1995. Improved plant regeneration from barley callus by increased copper levels. Plant Cell Tiss. Org. Cult. 43:267-269.

Lemaux, P.G., M-J. Cho, S. Zhang, and P. Bregitzer. 1999. Transgenic cereals: Hordeum vulgare L. (barley). In: I.K. Vasil (ed.) Molecular Improvement of Cereal Crops, pp. 255-316, Kluwer Academic Publishers

Zhu, H., L. Gilchrist, P. Hayes, A. Kleinhofs, D. Kudrna, Z. Liu, L. Prom, B. Steffenson, B. Toojinda, and H. Vivar. 1999. Does function follow form? Principal QTLs for Fusarium head blight (FHB) resistance are coincident with QTLs for inflorescence traits and plant height in a doubled-haploid population of barley. Theor. Appl. Genet. 99:1221-1232.