All-Union Inst. Plant Biotechnology

Isozyme patterns of inbred 346 somaclonal variants --E. E. Khavkin, M. I. Orlova, Z. B. Shamina and V. G. Chernysheva Regenerant plants (SC1) produced in callus culture from immature embryos of A654-derived inbred 346, as previously described (Chernysheva et al., Dokl. Akad. Nauk USSR 300:227, 1988), differed in several quantitative traits and manifested numerous morphological abnormalities eliminated in the SC2 generation. Two or three sibling SC2 plants were obtained from each of five SC1 plants with the following lineage: 1SC1 (3SC2 and 4SC2), 2SC1 (8SC2 and 10SC2), 3SC1 (1SC2 and 2SC2), 4SC1 (9SC2 and 11SC2) and 5SC1 (5SC2, 6SC2 and 7SC2). These SC2 varied from the initial plants in such traits as plant height, tassel length and the number of kernels per ear; particularly interesting was the significant diversity of sibs.

Several polyacrylamide and starch gel electrophoretic systems were employed to investigate acid phosphatase, alcohol dehydrogenase, esterase (E), glutamate-oxaloacetate-transaminase, isocitrate dehydrogenase (IDH), malate dehydrogenase, anodal and cathodal peroxidase (PRX) and 6-phosphogluconate dehydrogenase spectra in 3- to 5-day-old SC3 seedlings. While the seedlings of the control batch of inbred 346 exhibited uniform isozyme patterns, distinct alterations were found in two of ca. 20 analysed loci in at least seven of eleven investigated somaclonal variant seedlings.

Two most characteristic additional bands were apparent in anodal esterase patterns of scutella (Fig. 1). Their mobility as related to E8 band was 0.99 and 0.89 in lithium borate//lithium borate - Tris-citrate starch gel system and 0.89 and 0.78 in PAGE after Davis. By the former data we may tentatively identify these bands with E5 electromorphs 190 and 340 as described by MacDonald and Brewbaker (Hawaii Agr. Exp. Sta. Tech. Bull., No. 89, 1975). It is noteworthy that the faster of these two bands (340?) was also present in the mesocotyl pattern both in the control and somaclones. In neutral PAGE system modified after Taber and Sherman (Ann. N. Y. Acad. Sci., 121:600, 1964) a single additional band with the relative mobility of 0.83 was found in scutella of somaclones. This band was also typical of mesocotyls.

Figure 1. Esterase patterns of control (c) and somaclone (1-11) scutella as resolved by PAGE after Davis (a), in starch gel (b) and by neutral PAGE system (c).  (Figure unavailable - see hard copy.)

The most prominent changes in anodal PRX pattern of SC3 mesocotyls were found in the zone with mobility of 0.2 relative to bromphenol blue marker (Fig. 2) which could be preliminarily assigned to PRX3 as described by Brewbaker and Johnson (MNL 46:29, 1972): in addition to a single band of the control pattern, one or two apparently new bands could be seen in this zone in three somaclones. Two more weakly stained electromorphs appeared: one in 4, 9 and 10 SC3 mesocotyl spectra, while the second band with mobility of 0.02 (presumably PRX7) was found only in 10S C3.

Figure 2. Peroxidase patterns of control (c) and somaclone (4, 9, 10) mesocotyls resolved by PAGE after Davis.  (Figure unavailable - see hard copy.)

Among several quantitative changes in isozyme patterns, variations of staining intensity of the ADH1 ADH2 band and the slowest IDH electromorph were particularly promising for further investigation.

Previous zymographic studies of somaclonal variation justified that single gene mutations of structural enzyme loci were exceptionally rare events (Brettell et al., Mol. Gen. Genet. 202:235, 1986). It seems more plausible to suggest that the changes in isozyme patterns reported here resulted rather from switching off some factors regulating organ-specific expression. The appearance of the mesocotyl-specific esterase band in the scutellar spectrum of several somaclonal variants as well as divergence of sibs both in isozyme patterns and morphological traits seem to support this suggestion.

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