The majority of studies on anthocyanin pattern formation in maize have been done in vivo by screening kernels collected from the field at various days after pollination (DAP). Such studies have contributed immensely towards the understanding of phenomena such as clonal development of the aleurone using somatic reversions of R-st (Coe, in Maize Breeding and Genetics, Walden, ed., pp. 447-459, 1978) and elucidation of the intra-tissue differentiation in maize aleurone through studies on R-nj pigmentation (Styles et al., Can. J. Genet. 19:111-117, 1977; Prasanna and Sarkar, MNL 67:86-87, 1993). However, to closely follow the sequence of events in a single kernel/group of kernels simultaneously, the in vivo approach has inherent limitations. To circumvent these, we have adopted an in vitro approach.

Immature kernels (4-6 DAP) were transferred to test tubes containing 20 ml of culture medium (medium composition as in Gengenbach, Crop Sci. 17:489-492, 1977) under aseptic conditions as blocks of 4-8 kernels each. The tubes were incubated at 25±1 C in the dark. Kernels from homozygous R-mb:cc ears showed formation of the characteristic mb:cc pattern. Pigment initiation in vitro (14-16 DAP) was comparable to that in vivo except for a slight delay of 1-2 days. In aleurone patterns like that of R-mb:cc, which are symmetric and location-specific, it is important to ascertain whether the final pattern is due to progression of stripes arising from different regions of the kernel that coalesce thereafter or the pattern is pre-programmed without dependence on progression and coalescence. On the basis of the observations on pattern formation in several in vitro grown kernels, we conclude that in R-mb:cc, pigmentation pattern follows the latter. Intensity of pigmentation was less on initiation, with a gradual increase over time without any alteration in the basic pattern. However, colored spots appearing at the base of the kernel were relatively late in onset compared to those on the crown. Besides kernels showing mb:cc pattern, colorless and self-colored kernels were also recovered.

In order to ascertain differences in pattern formation of R-mb:cc and R-nj, immature ears from R-mb:cc/R-nj selfed lines were also cultured. As observed in vivo, the Navajo pattern showed a clear delay in its onset (a minimum of 3 days) in comparison to that of R-mb:cc. The kernels showed either nj, mb:cc or both nj+mb:cc expressions as would be expected on selfing of a R-nj/R-mb:cc heterozygote. Through these in vitro cultures, we could also obtain mature, fully grown/differentiated kernels. This approach, coupled with image analysis, can serve as a powerful tool to analyze the events underlying the formation of anthocyanin patterns like that of R-mb:cc. 

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