Annual Progress Report on Malting Barley Research March, 2002

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DEVELOPING BARLEY TISSUE CULTURE SYSTEMS TO IMPROVE PLANT
REGENERATION AND REDUCE SOMACLONAL VARIATION
Lynn S. Dahleen and Phil Bregitzer
USDA-ARS, Fargo, ND and Aberdeen, ID
A concerted effort to optimize tissue culture conditions for Harrington and Morex
has resulted in significant improvements in plant regeneration from tissues of these
important cultivars (see previous AMBA reports; Dahleen and Bregitzer, 2002). In
addition, these media improvements are applicable to many other tested cultivars of
diverse pedigree, with plant regeneration increases averaging 15-fold that obtained on the
original media. Vigorous regeneration is critical to successful transformation of barley
cultivars, and the improved techniques have been used to recover transgenic plants from
several difficult-to-transform cultivars, including Morex (Bregitzer et al., 2000).
Previous systems were very genotype-dependent (Wan and Lemaux, 1994), and the use
of unimproved culture systems has confined many transformation efforts to amenable
cultivars such as Golden Promise. Major efforts to introduce antifungal protein genes into
elite malting cultivars are underway (Bregitzer, Dahleen, Lemaux, research in progress).
It is expected that direct transformation of six-rowed cultivars will assist in screening for
FHB resistance.
In addition to facilitating the transformation of elite cultivars, improvements in
plant regeneration may be associated with reductions in somaclonal variation (SCV). In a
study of three different types of barley tissues, it was found that plants regenerated from
highly differentiated, meristematic tissues (which have superior regeneration
characteristics) were agronomically superior to plants regenerated from relatively
undifferentiated, embryogenic cultures (Bregitzer et al., 2002). Preliminary information
regarding this study can also be found in our 1999 report to AMBA. These studies did
not, however, address the relative importance of increased differentiation, versus the
changes in media formulations that are critical to creating and maintaining the
meristematic tissues. Interestingly, although 6-benzylamino purine (BAP) is of critical
importance to the successful production and maintenance of meristematic tissues, it can
be used also in the less differentiated, embryogenic tissues that have been used to develop
our improved medium. Thus, it was of interest to determine if the inclusion of BAP at
low levels could have a role in reducing SCV,
Although we are pleased with our progress towards a genotype-independent, nonmutagenic
tissue culture and transformation system for barley, we wished to further
investigate potentially useful media modifications and their role in reducing SCV. Two
objectives were pursued in the last year:
1) Investigate the influence of silicon on plant regeneration. Briefly, the justification
for these experiments is that silicon is not considered an essential nutrient and is
routinely omitted from common media formulations despite evidence that silicon
has a major role in plant growth and development (Epstein, 1999).
2) Determine if plants regenerated from our improved tissue culture media were
agronomically superior to those regenerated from the original media.