We have reported that floury-a inbred embryos were insensitive to lysine plus threonine (LT) growth inhibition in vitro (Rapela, Rev. Fac. Agr. UNLP 56:17-26,1980; Plant Cell Physiol. 23:285-291,1982). Also, embryos of a particular normal red flint inbred line (BP) were partially insensitive to such inhibition (Rapela, Rev. Fac. Agr. UNLP 56:27-37, 1980; Phyton 41:63-66, 1981). Normal flint inbred embryos were sensitive to LT inhibition. We report here the behavior of cell cultures of these inbred lines and their hybrids to LT inhibition.
Cultures were initiated with immature (1-2 mm long) embryos in MS medium plus 2% sucrose and 1 ppm 2,4-D. After two subcultures in the dark the proliferated callus was divided into small pieces (about 50 mg each), and pieces were transferred separately to the MS medium with LT, 1 mM. Calli were screened for LT resistance after 45 days of culture in LT medium. Embryo cultures were initiated with mature embryos in MS medium with LT, 1 mM, and were screened after 14 days in culture (Table 1).
The behavior of the inbred genotypes was similar both in embryos and in cell cultures. If the embryos were insensitive, partially insensitive or sensitive to LT inhibition the cell cultures were also insensitive, partially insensitive or sensitive, respectively. However, the behavior of two hybrid genotypes was different between embryo and cell cultures. A x C and A x D cell cultures were partially insensitive to LT inhibition while embryo cultures were sensitive. Surprisingly, the genotype A x B was sensitive to the LT inhibition either in embryo or in cell cultures.
Up to now none of the hybrid, partially insensitive cell cultures regenerated plants. So, our observations are very difficult to explain. However P.S. Carlson (Cell Tiss. Cult. Tech. for Cereal Crop Imp. p. 407, 1983), has suggested that in the culture of heterozygous clones (like our A x C and A x D genotypes), a number of distinct and stable subcultures appear with the expression of only one of the two alleles (in our case the floury-a). Such a process, termed allelic assortment, could explain our results. We are now investigating the molecular phenotypes of the hybrid LT resistant cell cultures via SDS-gel electrophoresis.
Table 1. Behavior of embryo and cell cultures in LT 1 mM. I = Insensitive (root length 60 % of control for embryo cultures, or 60 % healthy calli after 45 days for cell cultures). PI = Partially Insensitive (root length between 20-59 % of control for embryo cultures or 20-59 % healthy calli after 45 days for cell cultures). S = Sensitive (No roots or necrotic calli).
Miguel Angel Rapela
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