Introduction

Most U.S. barley transformation efforts use the cultivar Golden Promise (GP) because its high rate of green plant regeneration makes recovery of transgenic plants relatively straightforward. Unfortunately, GP falls significantly short of current North American agronomic and malting quality requirements. Thus, development of a commercially-viable cultivar for North American markets utilizing transgenic GP as a parent will be less efficient, as compared to using cultivars that do not contain numerous undesirable traits that are characteristic of GP. We have done extensive research on improving green plant regeneration from Morex and Harrington, but while regeneration has been improved (Dahleen 1995; Bregitzer et al. 1998), transformation attempts using these cultivars have met with very little success.

Studies based on the responses of Morex and Harrington indicated that there were certain media component and culture technique modifications that significantly improved green plant regeneration from callus tissues of these two cultivars. Morex and Harrington were chosen for these studies because they are the current U.S. malting quality standards, but Morex was released 20 years ago and has been mostly replaced by newer 6-rowed cultivars with better agronomic performance, and Harrington is not well adapted to the Red River Valley growing conditions. Although these experiments have not been concluded, certain modifications have been well-established as beneficial for Morex and Harrington, particularly the use of elevated copper concentrations. The objective of this study was to determine whether these higher copper concentrations were beneficial for a wide range of elite and modern Midwestern germplasm, and to identify potentially highly-regenerable lines for future transformation work. The potential of transgenic barley to help combat Fusarium head blight made the Midwest breeding lines and cultivars our first priority. This paper reports the plant regeneration from tissue cultures of twenty cultivars and advanced breeding lines on standard and improved media.

Materials and Methods

Each breeding program selected four to six lines for testing. Lines were tested in three sets according to when they were received. Set 1 consisted of four lines from Jerry Franckowiak (North Dakota State University) and six from Don Rasmusson (University of Minnesota). Set 2 consisted of five lines from Mike Bjarko (Busch Agricultural Resources, Inc.), and Set 3 consisted of five lines from Rich Horsley (North Dakota State University). Our previous work with Morex indicated that there can be tremendous variability for green plant regeneration between single plant derived lines (SPDLs) of a cultivar, so five plants were tested for each breeding line or cultivar. Each plant was individually harvested for further testing. Set 1 was planted in the greenhouse on Nov. 24, 1997, set 2 was planted on Jan 23, 1998, and set 3 was planted on May 20, 1998.

Four immature embryos (2-3 mm in length) were placed in each petri plate, with ten plates per treatment per SPDL. Two treatments were used, standard MS CuSO₄ levels (0.1 microM) and high CuSO₄ levels (5.0 microM). Cultures were initiated on MS medium solidified with 3.5 g/L Phytagel, modified as described by Bregitzer (1992), and supplemented with 3% maltose and 4.5 mg/L 2,4-D. Plates were incubated in the dark at 22-25°C. After three weeks, half of the calli from three of the four embryos were transferred to fresh initiation medium, under the same conditions. After another three weeks, calli were transferred to maintenance medium, with the 2,4-D level decreased to 2 mg/L and 0.1 mg/L BAP added, and placed under dim lighting. Calli remained on this medium for six weeks (one transfer at three weeks), then were transferred to regeneration medium without plant growth regulators, and placed under fluorescent lights. Four weeks after transfer, vigorous seedlings with both root and shoot development were counted, and remaining shoots were transferred to rooting medium. This medium consisted of half-strength modified MS (as described above) without growth regulators, with 3% sucrose substituted for the maltose. After four weeks on rooting media, vigorous seedlings with both root and shoot development were counted, and the two plant counts were totaled to determine the number of green and albino plants per plate. All media were autoclaved in three parts as described by Bregitzer et al. (1998). Morex SPDL2, the line used for culture improvement studies, was included in all sets for comparison.

Levels of contamination in some genotypes were very high, especially in MNS93, ND15403-3, and B1202 which had 50% or more cultures contaminated. The bacteria caused calli to brown early, but didn't spread to the medium until transferred to the light for regeneration. We normally have very low levels of contamination, and other cultures from the sets started on the same day showed little or no contamination, possibly indicating some sort of internal contamination of the seed or high level of undetected damage by thrips carrying bacteria. We routinely remove visibly damaged seed before surface sterilization.

Results

The inherent variability of regeneration from tissue cultures of barley is shown in this study by the differences in performance of Morex SPDL2 in the three sets. Regeneration from this line varied from 11.4 green plants/plate in set 1 to 35.8 green plants/plate in set 3 (Tables 1-3). These differences are likely due to different light levels in the greenhouse during growth of the plants used as embryo sources (Dahleen 1999). Sets 1 and 2 were planted during the winter months, when day lengths are short in North Dakota, while set 3 was planted in the spring when day lengths are much longer. Because of this variability due to planting date of the embryo donor plants, sets cannot be directly compared to each other.

Culturing on media containing increased copper concentrations increased regeneration from each cultivar or line (Tables 1-3). All lines regenerated some green plants on high copper concentrations, including those 6-rowed lines that did not regenerate on media containing standard copper concentrations. Mean albino frequency was low on both high and standard copper concentrations, ranging from 0 to 1.8 albino plants/plate. Differences in green plant regeneration on high copper concentrations between lines were huge, ranging from 0.6 green plants per plate for MN93-53 up to 42.8 for Logan. As is commonly observed in North American genotypes, the 2-rowed lines tend to regenerate many more plants than the 6-rowed lines.

Considerable variation was observed between SPDLs within a cultivar or breeding line (Tables 4-6), likely due to a combined effect of normal tissue culture variability and small genetic differences between seed. Some lines showed consistently high levels of regeneration, like Logan and B1201, while others gave consistently low levels of regeneration, like Robust and MN93-53. Within each set, there are several SPDLs that look promising as transformation candidates. From set 1, several SPDLs from Logan and Conlon and single SPDLs from ND16050, ND15403-3, MNBrite, and MN93-67 look promising. SPDLs worth further examination from set 2 included some from B1202, Merit and B2601, and from set 3 included ND15614, ND15477 and ND15422. Any of these lines would be a better starting point than GP from an agronomic perspective.

References:

Bregitzer, P. 1992. Plant regeneration and callus type in barley: effects of genotype and culture medium. Crop Sci. 32:1108-1112.

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

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

Dahleen, L.S. 1999. Donor-plant environment effects on regeneration from barley embryo-derived callus. Crop Sci. 39:In Press (May-June issue).