Cotyledonary explants of 4-day-old Cucumis melo cv. 'Hale's Best Jumbo' in vitro seedlings showed maximum initiation of shoot buds when cultured onto a revised Murashige & Skoog medium supplemented with 5/~M indole-3-acetic acid and 5/~M benzylaminopurine and cultured at 25-29°C under low light intensity (5-30#molm-2s-~). Subculture of the shoot buds onto the same medium without auxin and supplemented with 3 #M benzylaminopurine caused the development of shoots from 30% of the buds. The presence of abscisic acid significantly increased the number of explants producing shoot buds. Bud initiation was affected by genotype, seedling age, light intensity, and temperature. Addition of gibberellic acid, thidiazuron or silver nitrate to regeneration medium did not improve either bud initiation or shoot regeneration.
SUMMARYSoybean protoplasts, isolated from liquid suspension culture, were successfully infected with cowpea mosaic virus (CPMV) and with southern bean mosaic virus (SBMV). Poly-L-ornithine (PLO) was required for infection with either virus. As detected by fluorescent antibody, 70 to 9o % of the protoplasts were infected by CPMV when the inoculation medium contained o'4 M-sorbitol, o'5/~g virus/ml, 2"5/zg PkO/ml, IO raM-potassium phosphate buffer, pH 6.3, and o'5 mM-CaCI~, and inoculation was performed at 23 °C. This maximum level of infection was decreased sixfold when CaC12 was omitted. Inoculation at temperatures below l o °C also decreased infection substantially. With SBMV a maximum of 3o to 35 % of the protoplasts were infected when o'4 M-sorbitol, 2 to 2"5/~g virus/ml, 2 #g PLO/ml, ro mm-tris-HC1 buffer, pH 8.o, I mM-MgSO4 and 0. 5 mM-CaC12 were present in the inoculum. Less than 5% of the protoplasts were infected when magnesium and calcium salts were omitted. The major advantage of the use of soybean cell suspension culture for plant virus studies is that it can provide a reliable and continuous source of cells for protoplast isolation. These soybean protoplasts allow synchronous infection and replication under sterile conditions without antibiotics and also a high degree of reproducibility.
Haploid tobacco (Nicotiana sylvestris) plants were inoculated with a yellow strain of tobacco mosaic virus (TMV-Flavum) and then exposed to 500 rads of acute gamma radiation. Leaf strips cultured on callus-inducing medium yielded two types of colonies: 1) yellow, virus-infected and 2) green, apparently healthy. Of the 3210 calli scored, approximately 5% were virus-free, and after regeneration, 0.2% were resistant at the plant stage. Later, adult plants, both TMV-resistant and TMV-susceptible, produced self-fertile, diploid flowers. Both seedling progeny and rooted cuttings from resistant stock plants showed resistance to TMV infection. This resistance was characterized by restricted virus multiplication and movement within the infected plant resulting in a 3-8 week delay in symptom expression.
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