Application of plant tissue and organ culture in soybean improvement for resistance to Septoria glycines

Abstract

Culture filtrate of Septoria glycines Hemmi grown in Septoria medium contained a host-specific toxin which induced typical symptoms of brown spot disease on soybean leaves. Culture filtrate from 21-day-old cultures caused chlorosis on detached cotyledons and inhibited the growth of soybean callus. Less than 20% of the seeds germinated on culture filtrate-containing water-agar at a 1:2 dilution. Embryogenesis was stimulated when germinated soybean seeds on water-agar (6 g/l) containing 40 $\mu$M 2,4-dichlorophenoxy-acetic acid (2,4-D) were transferred to Murashige and Skoog (MS) medium containing 20 $\mu$M benzylaminopurine (BAP). Organogenesis was facilitated by transferring organogenic callus from MS medium with 20 $\mu$M BAP to MS medium with 1 $\mu$M BAP. Plants resistant to the pathotoxic culture filtrate of S. glycines were regenerated from immature embryo of soybean cultivar (cv.) BSR201 and from cotyledonary nodes of mature seeds of the cvs. BSR201, Fayette, and one breeding line, L1615. The incubation period for S. glycines in progeny of resistant plants (cv. BSR201) obtained from immature embryo was five weeks longer than the incubation period in susceptible plants. The resistant plants were small, but not all small plants were resistant. Variation was also observed in plant maturity, growth habit, and fertility.

A host-specific pathotoxin isolated from the culture filtrate of S. glycines is an autoclave-stable, high molecular weight (approximately 20,000 dalton) uronic acid-containing polysaccharide. The toxin causes typical disease symptoms of brown spot on cotyledons and leaves of soybean. This toxin was purified by sequential CM-cellulose treatment, DEAE-cellulose chromatography, dialysis, gel filtration, and 5% charcoal treatment. Drying the toxin under flash-evaporation at 46$\sp\circ$C in vacuo destroyed over 99% of the toxin activity. Treatments with 1 N HCl at 90$\sp\circ$C for 3 hr or with proteinase K (5%) did not abolish the toxicity. Oxidation with 35 mM IO$\sb4\sp-$, incubation with $\alpha$-mannosidase (38 units/ml), or $\beta$-galactosidase (1 unit/ml), or $\beta$-glucosidase (16 units/ml) all markedly reduced toxin activity.