Endogenous genetic regulation of anthocyanin production in embryogenic callus

--John P. Bodeau and Virginia Walbot

Embryogenic (ìtype IIî) maize callus cultures are generally colorless, but calli of certain genotypes will synthesize anthocyanin either in response to light (G. Dietrich, G, unpublished data), or following particle-gun introduction of constitutively expressed R and C1 gene-family members (Goff et al., EMBO J 9:2517). We want to better understand the endogenous genetic regulation of anthocyanin pigmentation in embryogenic callus, with two primary goals in mind. 1) By determining which tissue-specific regulatory alleles enable pigment synthesis, we may learn which natural tissue is most closely related to embryogenic callus. 2) After we know the regulatory alleles required for anthocyanin synthesis in callus, we may initiate embryogenic lines lacking a single structural or regulatory gene, in theory allowing any of these genes to be used as a visual marker for transformation.

We began in 1989 by crossing several pigment tester lines to the inbred line A188, from which embryogenic callus is easily initiated. A188 has the regulatory genotype r-r c1 b pl, with wild-type alleles of the structural genes C2, A1, A2, Bz1, Bz2, and Pr. Thus it is colorless in all tissues except for pale pink anthers and red seedling leaf sheaths; embryogenic A188 callus is colorless under all growth conditions we have tested, including light and cold treatments. The alleles introgressed included R-r, R-g, R-nj, R-scm2, r-g, B, B-peru, C1, C1-S, C1-I, Pl, c2, a1, a2, bz1, bz2, and an-bz2-6923. These alleles came from a variety of backgrounds, predominantly W23 and K55, which are virtually incapable of giving rise to embryogenic callus. By a program of repeated backcrosses to A188, we have produced several genotypes having from 50% to 87.5% A188 nuclear background, from which type II embryogenic calli were initiated in August, 1991.

Lines with a higher fractional component of A188 were significantly more efficient at initiating type II embryogenic callus (Table 1). The identity of the introgressed background also affected initiation efficiency: K55-containing material was relatively inefficient, while W23-containing lines were at least as efficient as pure A188. Our attempt to improve the callus-induction efficiency of desired genotypes by a backcrossing program to A188 was thus successful.

Table 1 Percent of immature embryos which initiated embryogenic type II callus as a function of introgressed background.
 
Fraction A188: 1.0 .875 .75
A188 inbred 42% (n=36)    
W23   48% (n=205) 42% (n=115)
K55   35% (n=48) 0% (n=25)

Pigment production in embryogenic calli was scored after growth for two weeks in near darkness, or after a two hour exposure to full sunlight followed by two weeks' growth under low wattage fluorescent bulbs (Table 2). Because of ongoing genetic segregation, sampling error, and relatively mild light treatments, the possibility of false negatives is quite likely, and must be considered when interpreting all observations. When it was seen, pigment was usually in the surface cells of the undifferentiated, friable mass forming the bulk of embryogenic callus. The embryoids themselves, however, were colorless, even when arising from red callus. In only one genotype (JD40), containing the B-peru allele, pigment was seen in the suspensor-like part of embryoids. However, calli of this genotype were unlike normal type II calli, and grew poorly.

Pigment accumulated in both dark- and light-grown calli of the genotypes R-r C1 B Pl (JD26, JD30) and R-r C1 b Pl (JD28), while light-dependent pigment accumulation occurred in both R-r C1 B pl (JD27) and R-r C1 b pl (JD29). Thus, on the basis of positive scoring of pigmented calli, we can definitely state that B is not required for pigment synthesis, while Pl is probably required to eliminate a light-requirement. Our observations match Racchi's (Plant Cell Rep. 4:184) observations in non-embryogenic endosperm calli of K55/W23 hybrid background. All of his material, of genotype R-r C1, and differing only in its B pl genotype, eventually made anthocyanin, but only Pl genotypes made it rapidly (e.g., within 30 days after culture). Thus neither B nor Pl were required in endosperm callus, but Pl was an important enhancer of pigmentation.

Table 2. Scoring of embryogenic callus lines initiated in 1991: genotype, background, and pigmentation phenotypes.
 
    %A188/ Color: dark Color: light
Cross  Genotype Other bkgd.    
Regulatory genotypes        
JD1(x) r-r c1 b pl 1.00 cl cl
JD19 x 1 R-g/r-r C1/c1 b pl .50/WKN1 cl cl
JD26.1-1(x) [R-r/r-r Cl/? Pl/? B/?] (x)  .875/W23 Red Red
JD26.2-2(x) R-r/R-r [C1/? B/? Pl/?] (x) .875/W23 Red Red
JD27.2-1(x) pl [R-r/r-r C1/? B/?] (x)  .875/W23 cl Red
JD28.1-2(x) R-r/R-r b [C1/? Pl/?] (x)  .875/W23 Red Red
JD29.1-1(x) R-r/R-r b pl [ C1/?] (x)  .875/W23 cl Red
JD29.1-3(x) b pl [R-r/r-r C1/c1] (x)  .875/W23 cl cl
JD30.1-1(x) R-r/R-r [C1/c1 Bwk/? Pl/?] (x) .875/K55 Red Red
JD30.1-4(x) pl [R-r/r-r C1/c1 Bwk/?] (x)  .875/K55 cl Red
JD31.1-2(x) R-r/R-r b [C1/c1 Pl/?] (x) .875/K55 cl cl
JD32.1-5(x) r-r pl [C1/c1 B/?] (x) .875/W23 cl  cl 
JD34.2-1(x) r-g b pl [C1/c1] (x) .75/W23 cl cl 
JD35.1-1(x) r-g C1/C1 b pl .75/W23 cl  cl 
JD37.1-2(x) R-r/R-r c1 [B/? Pl/?] (x) .75/W23 cl \\ 
JD40.1-3(x) r-? [C1/? BPeru/? Pl/? ] (x) .75/? cl (Red suspensors) cl (Red suspensors)
JD40.1-6(x) r-? C1/C1 Bperu/Bperu pl 75%/? cl \\ 
JD41.1-1(x) b pl [R-r/? C1-S/c1] (x) .875/? cl cl
Structural genotypes        
JD42.1-3(x) a1 R-g/R-g b pl [C1/c1] (x)  .875/KN1 cl cl
JD43.1-5(x) a2 R-g/R-g C1/C1 b pl .75/? cl cl
JD46.2-4(x) bz1 R-g/R-g C1/C1 B/B [Pl/?] (x) .75/W23 cl (Bronze) cl

The R-r and C1 requirements are more ambiguous, as we must contend with negative evidence. C1 is probably required for callus pigmentation, as c1 R-r B Pl (JD37) calli were colorless. This conclusion is uncertain, however, because only two calli were scored, and these grew poorly. As for R, in an earlier experiment it appeared that the seed-component (R(S)) is not required for callus pigment synthesis, because r-r B C1 pl calli left for several days on a lab bench in diffuse sunlight did indeed accumulate pigment (data not shown). Under the light conditions in the current experiment, however, r-r B C1 pl calli (JD32) failed to make pigment in the dark or in the light. Meanwhile, the plant component of R (R(P), or -r) may be required, but is not sufficient for pigmentation. R(P) is the only permissive regulatory allele that inbred A188 (r-r b c1 pl) has, and A188 calli are always colorless. Experiments are in progress to better establish the R-r and C1 requirements, which will give us additional insight into the relatedness of embryogenic callus to other plant tissues.

In addition to genotypes with varied regulatory genotypes, we have successfully initiated callus lines homozygous recessive for the structural genes a1, a2, and bz1. All of these calli were colorless, although the exact regulatory genotypes of these lines are uncertain. Experiments are in progress to verify that these calli have permissive regulatory genotypes. If so, we will attempt to reintroduce the missing genes by particle bombardment, and visually select for pigmented, i.e. transformed, tissue.

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