Biotechnology Applications of Plant Callus Cultures

We have developed an Agrobacterium mediated transformation procedure for sugarbeet, which is known for its recalcitrant behavior in vitro. A friable type of callus was used as the starting material, and combinations of different chimeric gene constructs, consisting of antibiotic or herbicide resistance genes, were evaluated. Sugarbeet was engineered for resistance to the broad spectrum herbicide glufosinate-ammonium or to sulfonylurea compounds by introducing either the bialaphos resistance gene (bar) or genes encoding mutant acetolactate synthases (ALS). Field grown progeny of transformants expressing the bar gene were resistant to commercial levels of glufosinate-ammonium applied in the field. Transformants expressing a mutant ALS gene were resistant to field levels of sulfonylurea compounds applied under greenhouse conditions. The procedure described proved to be applicable for lines with different genetic backgrounds.

Transformation of Sugarbeet (Beta vulgaris L.) and Evaluation of Herbicide Resistance in Transgenic Plants

Plantlets were regenerated by adventitious shoot budding in tissue culture from leaf explants of a single genotype of sugar beet. DNA was extracted from the parental plant and from 120 regenerants. RAPD analysis was carried out using five decanucleotide primers; 4,557 RAPD marker bands were examined and two polymorphisms were observed. Thirty secondary regenerants were then derived, using the same tissue culture technique, from thirty of the primary regenerants. Again RAPD analysis was employed and a single band polymorphism was observed out of 1,050 bands examined. The overall frequency of detection of somaclonal polymorphisms using RAPD (3 in 5,607 = 0.05%) is similar to frequencies previously reported using isozyme and RFLP technologies.

The detection of somaclonal variants of beet using RAPD.

Six sugarbeet (Beta vulgaris L.) lines (GWI-248, SPB-11, MonoHy 55, SMS-1, EL45 and FC607) were tested for regeneration. Shoot cultures were initiated in vitro from naked, sterilized embryos obtained from mature seed. Excised petioles from cultured shoots were plated on Gamborg's B5 medium and four modified Murashige and Skoog (MS) media. A medium containing MS inorganic salts supplemented with 0.4 mg/1 N6-benzyladenine, 0.1 mg/1 indole-3-butyric acid, ten vitamins and six amino acids, termed RV, was superior for both adventitious shoot and callus formation. Callus was observed only on RV medium and only on petioles that did not develop adventitious buds directly. Rooting of regenerated shoots and development of complete plants was accomplished by transfer to Gamborg's B5 medium with 5 mg/l indole-3-butyric acid as the sole phytohormone. The complete process of regeneration through adventitious shoot production took from 4 to 6 weeks from explants to rooted plants. The callus that formed on nonorganogenic petioles was regenerative when transferred to fresh RV medium. Regeneration from callus occurred mainly by shoot organogenesis but also by somatic embryogenesis at a low frequency.

An improved medium for adventitious shoot formation and callus induction in Beta vulgaris L. in vitro

It has been known for over 25 years now that plant cell and tissue cultures undergo genetic erosions and show changes of various types, especially in chromosome numbers and ploidy level (Partanen 1963; D’Amato 1965; Murashige and Nakano 1966). However, till the mid 1970’s such changes were considered undesirable and were therefore discarded because the main emphasis was on clonal propagation and genetic stability of the cell cultures. Extensive studies conducted during the last decade have shown that the cell cultures, especially on periodical subculturing, undergo various morphological and genetic changes, i.e., polyploidy, aneuploidy, chromosome breakage, deletions, translocations, gene amplification, inversions, mutations, etc. (see Nagl 1972; Meins 1983; D’Amato 1985). In addition, there are changes at the molecular and biochemical levels (Day and Ellis 1984; Landsman and Uhrig 1985; Ball and Seilleur 1986) including changes in the DNA (Berlyn 1982; Cullis 1983), enzymes (Brettell et al. 1986b), gliadin (Cooper et al. 1986), etc. Since the publication of Larkin and Scowcroft (1981) there has been an upsurge of interest, and the attitude toward these changes has shifted to using the somaclonal variations for plant improvement; they are intentionally induced and much sought after.

J.W. Saunders

Papers

One Step Shoot Regeneration from Callus of Whole Plant Leaf Explants of Sugarbeet Lines and a Somaclonal Variant for in vitro Behavior