Effects of HC toxin on growth and regeneration of susceptible and resistant callus

Helminthosporium carbonum, race 1, a fungal pathogen of corn, produces a host-specific toxin (HC toxin) known to be a pathogenicity factor for the disease Helminthosporium leaf spot. The toxin has been purified and the structure is known (J. Walton and E.D. Earle, Biochem. Biophys. Res. Comm. 107:785). Major host resistance and insensitivity to the toxin are conditioned by a single dominant gene Hm (O.E. Nelson and A.J. Ullstrup, J. Hered. 55:195). Root-growth assays have shown that certain corn genotypes susceptible to the fungus are sensitive to low toxin concentrations (root growth is inhibited). Corn genotypes resistant to the fungus are also sensitive to the toxin, but at higher concentrations. We describe here studies which demonstrate a differential effect of HC toxin on growth and regeneration of susceptible and resistant callus and our attempts to recover novel resistant genotypes using an in vitro selection system.

Our initial attempts to establish regenerable callus of susceptible and resistant near-isogenic inbred lines, Pr and Pr-1, resp., were unsuccessful. Therefore, to obtain susceptible genotypes which were amenable to tissue culture, we used the following approach. The susceptible inbred Pr (hm hm), which does not produce regenerable callus, was crossed to the resistant inbred W182BN, which readily produces regenerable callus. The resulting F1 progeny were self-pollinated. Plants derived from those self-pollinations were grown in the field and inoculated with a spore suspension of H. carbonum race 1 to identify susceptible genotypes. A F2 population of that cross segregated 136 resistant to 39 susceptible plants, which was an acceptable fit to the expected 3 resistant:1 susceptible ratio if the Hm locus were segregating. In 1985 and 1986, immature embryos from susceptible and resistant plants were excised ten days after pollination and plated onto a maintenance medium containing 15 µM 3,6-dichloro-o-anisic acid (D.R. Duncan, M.E. Williams, B.E. Zehr and J.M. Widholm, Planta 165:322). Resistant and susceptible callus was subcultured at three-week intervals. Plants were regenerated by transferring callus onto a modified LS medium with no hormones and decreasing percentages of sucrose (10, 4 and 2 percent - two to four weeks on each).

The toxin preparation used in these studies was kindly provided by Dr. V. Macko at the Boyce Thompson Institute for Plant Research, Cornell University, Ithaca, NY. Toxin was incorporated into the medium at the appropriate concentrations just before the media were poured into plates.

To determine toxin concentrations inhibitory to callus growth, susceptible and resistant calli were placed on media containing concentrations of 0, 0.5, 1, 2, 5, 10, and 20 µg/ml toxin. The callus used in those experiments was not friable and had many organized areas. Initial and final (4 weeks later) fresh weights of the calli were recorded. The percent increase in growth for each treatment was calculated (Figure 1). Exposure to increasing toxin concentrations gave progressively greater growth inhibition, as well as amount of necrotic tissue (data not shown) of susceptible callus. Resistant callus was not inhibited at concentrations up to 20 µg/ml (Figure 1) but was inhibited at the concentration of 50 or 100 µg/ml (Figure 2).

To study toxin effects on regeneration 16 susceptible and resistant calli were plated onto regeneration media with 0, 1, 2, 5, 10 and 20 µg/ml toxin (Table 1). At 5 µg/ml and higher, susceptible callus formed no large shoots, whereas resistant callus formed large shoots at all toxin levels.

Table 1. Organogenesis on Susceptible(S) and Resistant(R) Callus with Various Levels of HC Toxin.
Toxina µg/ml
# calli w/ small shoots
# calli w/ roots
# calli w/ large shoots
total large shoots
  S R S R S R S R
        15 14 5 W 17
1 1 0 9 1 0 16 10 7 14 4
2 11 14 10 1 3 5 2 1 0 6
5 8 11 1 13 0 7 0 :0
10 5 11 1 11 0 3 0 9
20 1 8 0 9 0 3 0 9

aSixteen calli were on regeneration medium (10 and 4% sucrose) containing the toxin concentration indicated for four weeks.

Those data were used as the basis for the following selection scheme. Objectives were to recover novel resistant genotypes and compare the efficacy of mutagens, age of callus and selection pressure for the recovery of resistant genotypes. A total of 5,676 calli (10 to 20 mg) were used. The mutagens sodium azide and ethyl methane sulfonate were applied to callus at predetermined sublethal concentrations prior to toxin exposure. Callus which had been in culture for 3 months (established 1985) and 15 months (established 1986) was subjected to toxin selection. Initial toxin concentrations of 2, 5, and 10 µg/ml were used. Those calli on 2 µg/ml were subjected to a stepwise toxin increase of 5 µg/ml at the first transfer. Some of the calli exposed to 5 µg/ml were subjected to 10 jig/ml at the first transfer. At each selection or transfer (approximately 3-week intervals) individual calli were scored for percent necrosis. All nonnecrotic areas were transferred. The percentages of calli (based on initial number of calli) which were eliminated in each selection regime for each of six selection cycles are shown in Figure 3. To date, we have not recovered any resistant callus in any selection regime. Mutagen application or varying callus age did not affect the recovery of resistant callus. Growth of all susceptible calli was inhibited by the toxin and all eventually became 100% necrotic. Callus that we knew to be resistant (W182BN) was exposed to similar toxin levels for the same amount of time. That callus readily grew and regenerated resistant plants at the fourth, fifth, and sixth selection cycles. Failure to recover resistant callus might be due to an inability to identify resistant cells within relatively slow-growing callus cell populations. Moreover, susceptible cells surrounding a resistant cell might leak components which are lethal to the resistant cell. To address those possibilities, additional selection experiments using fast-growing cell suspensions are underway.

Figure 1. Percent increase in fresh weight of susceptible and resistant callus exposed to various concentrations of HC toxin for four weeks.

Figure 2. Final fresh weight and percent necrosis of resistant callus exposed to various concentrations of HC toxin for four weeks. Initial fresh weight of individual calli was 10 mg.

Figure 3. Percent calli eliminated in various selection regimes during six cycles of selection. Paired numbers in legend refer to toxin exposure (µg/ml) of the first selection cycle followed by toxin exposure of the subsequent selection cycles (2 to 6).

S.J. Wolf and E.D. Earle

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