Maize anthocyanin genotypes that efficiently give rise to friable, Type II embryogenic callus were produced by repeated backcrossing to the inbred line A188. These lines were produced to better understand the endogenous genetic regulation of anthocyanin synthesis in such callus and to facilitate use of the anthocyanin genes as visible markers for transformation. These lines, summarized in Table 1, are available upon request and have been submitted to the Stock Center. The overall morphology of the backcrossed lines resembles inbred A188 plants, which under summer conditions at Stanford typically are short in stature, thick stemmed, rarely tillered, early flowering, and form kernels with a pronounced sharp point at the silk attachment site. Immature embryos of most of the lines efficiently initiated embryogenic callus when plated on N6 media.

Anthocyanin accumulated in callus of several genotypes. When present, anthocyanin accumulated in the surface cells of undifferentiated, friable tissue forming the bulk of Type II callus. The embryoids arising from red callus, however, were colorless, although the suspensor-like supporting stalk was often pigmented. Prolonged light treatment increased anthocyanin accumulation in all pigmented genotypes, and was absolutely required for pigmentation in pl calli.

As in other tissues, both an R-family member (R or B) and a C1-family member (C1 or Pl) were required for callus pigmentation. Multiple R-family alleles conferred pigmentation. The genotype r-g b C1 pl, lacking any functional R or B allele, was colorless, indicating that at least one R-family member is required in callus. Both the S (seed) or the P (plant) component of the complex R-r locus were functional in callus. R-g calli, lacking the plant component, R(P), and r-r calli, lacking the seed component, R(S), were both pigmented. R-r genotypes were, of course, also strongly pigmented. An additional R allele, R-navajo (R-nj), was also functional in callus. R alleles R-scm2 and r-cherry:Hopi, and B alleles B-Intense and B-peru, were not required in addition to a functional R allele as discussed above. Type II calli of the correct genotypes were not successfully initiated to test whether these alleles were individually sufficient.

The requirement for a C1-family member resembled that of mature plant tissues: either Pl or pl, plus prolonged light treatment, were necessary and sufficient for callus pigmentation. C1 is not required, nor is it sufficient for callus pigmentation in the dark in the genotype R-r B C1 pl, or any other. Interestingly, light-induced pigmentation was seen in the genotype R-g b C1 pl, which in planta accumulates anthocyanins independently of light, but only in aleurone tissue.

In addition to regulatory genotypes, we initiated callus lines individually homozygous recessive for the structural genes a1, a2, bz1, and bz2. None of these callus lines accumulated visible anthocyanin, but some turned brownish or necrotic more quickly after subculture than did wild-type calli. These observations suggest that flavonoid intermediates accumulated in calli, much as they do in plant tissues.

While embryogenic callus pigmentation has similar genetic requirements as intact plant tissues, normal patterns of tissue-specific gene expression are not maintained in callus. The R-family alleles R-nj, R(S), and R(P) were each functional in embryogenic callus; in planta R(S) and R(P) act in mutually exclusive seed or plant tissues, while R-nj acts in both. For the C1 requirement, either Pl or pl plus prolonged light treatment, but not C1, was sufficient. Pigmentation conferred by the combination of R(S) and pl plus light (genotype R-g b C1 pl) was unexpected because R(S), normally active only in the aleurone, in this case required either light-induced expression of pl, which is not usually active in aleurone, or light-induced (enhanced?) expression of C1, which is strongly active in aleurone in the dark. Thus either alleles that function in mutually exclusive aleurone and somatic tissues are co-expressed, or the wild type C1 allele acquires a light requirement in callus. In any case Type II callus appears to represent a novel tissue-type with similarities to both seed and plant tissues. 

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