An approach towards genetically engineered cell fate mapping in maize using the Lc gene as a visible marker: transactivation capacity of Lc vectors in differentiated maize cells and microinjection of Lc vectors into somatic embryos and shoot apical meristems

To establish a system for genetically engineered cell fate mapping, different vectors carrying the Lc gene, a member of the R gene family, were delivered into embryonic and meristematic cells of maize by the microinjection technique. Vectors in which the Lc cDNA is driven either by a constitutive promoter (CaMV 35S), with or without the Adh1 intron 1 of maize, or a tissue-specific promoter (phosphoenolpyruvate carboxylase, PEPC) as well as self-replicating wheat dwarf virus (WDV) vectors carrying a Lc-expression-cassette, have been tested. The ability of these vectors to transactivate was evaluated in mesophyll-derived protoplasts of the maize genotype appropriate for these microinjection experiments. The expression product of the introduced Lc gene can substitute for mutated R and B loci, resulting in anthocyanin production. Analogous results were obtained by microinjection into organized tissues, where transactivation of anthocyanin biosynthesis resulted in pigmented sectors in somatic embryos (B79) and in the leaves of plants regenerated from the cultivated shoot apical meristems (K55, r-g, b). The tissue-specific appearance of pigmented sectors in leaves, using the mesophyll-specific PEPC promoter suggests the possibility of using this approach for layer-specific cell fate studies. The presence of the introduced plasmids in leaves showing red sectors 20–30 days after injection was proven by PCR analysis.     

Anthocyanin accumulation enhanced in <Emphasis Type="Italic">Lc</Emphasis>-transgenic cotton under light and increased resistance to bollworm

Breeding of naturally colored cotton fiber has been hampered by the limited germplasm, an alternative way is to use transgenic approach to create more germplasm for breeding. Here, we report our effort to engineer anthocyanin production in cotton. The maize Lc gene, under the control of the constitutive 35S promoter, was introduced into cotton through genetic transformation. Our data showed that the expression of the Lc gene alone is sufficient to trigger the accumulation of anthocyanin in a variety of cell types including fiber cells in cotton. However, the accumulation of colored anthocyanin in cotton fibers requires the participation of light signaling. These data indicate that it is feasible to engineer colored fibers through transgenic approach in cotton. Furthermore, we showed that the Lc-transgenic cotton plants are resistant to cotton bollworm. These transgenic plants are, therefore, potentially useful for cotton breeding against cotton bollworm.     

Lc as a non-destructive visual reporter and transposition excision marker gone for tomato

The maize anthocyanin regulatory gone Lc gives rise to greatly enhanced anthocyanin accumulation when introduced into tomato. Under the control of the cauliflower mosaic virus promoter Lc was seen to mediate enhanced pigmentation in all vegetative tissues, including roots, under high light (or sunlight) conditions. This paper demonstrates that the gone can be used as a powerful non-destructive cell autonomous visual excision marker which will provide a valuable tool for transposon mutagenesis, the study of transposon biology and for studying cell lineages at the whole-plant level.     

Expressing the maize anthocyanin regulatory gene Lc increased flavonoid content in the seed of white pericarp rice and purple pericarp rice

The colour of red, purple, brown and white occurs in pericarp of rice. Here, the maize anthocyanin regulatory gene Lc under control of the promoter of the rice glutelin gene Gt1 was introduced in the white pericarp rice “Chao2-10” and purple pericarp rice “Qingjiaozidao”. The results demonstrated that some transgenic “Chao2-10” rice pericarps became brown, and the total flavonoid contents in the unpolished rice of the two transgenic rices increased significantly compared with their respective controls. Unpolished rice kernel thickness and weight in the two transgenic rices decreased slightly.     

Overexpression of maize anthocyanin regulatory gene Lc affects rice fertility

Seventeen independent transgenic rice plants with the maize anthocyanin regulatory gene Lc under control of the CaMV 35S promoter were obtained and verified by molecular identification. Ten plants showed red spikelets during early development of florets, and the degenerate florets were still red after heading. Additionally, these plants exhibited intense pigmentation on the surface of the anther and the bottom of the ovary. They were unable to properly bloom and were completely sterile. Following pollination with normal pollen, these plants yielded red caryopses but did not mature normally. QRT-PCR analysis indicated that mRNA accumulation of the CHS-like gene encoding a chalcone synthase-related protein was increased significantly in the sterile plant. This is the first report to suggest that upregulation of the CHS gene expression may result in rice sterility and affect the normal development of rice seeds.     

Displaced and tandem duplications in the long arm of chromosome 10 in maize

Lc, an anthocyanin pigmenting factor mapping somewhat more than one unit distal to R, is borne on a chromosomal segment which is homologous with part of the R-r:standard duplicated segment. Deficiencies and tandem duplications of the R to Lc region arise from exchanges within these obliquely paired homologous segments. The deficiencies are transmitted with a high, although reduced, frequency by the male gametophyte and are homozygous viable. Yet, the R to Lc region is not duplicated either proximal to R or distal to Lc. Thus the Lc-marked segment and either the P- or the S-marked segment of R-r constitute a displaced duplication. Such an arrangement can initiate a tandem and displaced duplication cycle.———No evidence was obtained for fractionation of the compound phenotype conditioned by Lc.     

Comparison of constitutive promoter activities and development of maize ubiquitin promoter- and Gateway-based binary vectors for rice

In transgenic experiments, we often face fundamental requirements such as overexpressing a certain gene, developing organelle markers, testing promoter activities, introducing large genomic fragments, and combinations of them. To fulfill these multiple requirements in rice, we developed simple binary vectors with or without maize ubiquitin (UBQ) promoter, Gateway cassette and fluorescent proteins. First, we compared stabilities of cauliflower mosaic virus 35S and maize UBQ promoters for constitutive gene expression in transgenic rice. We show that the 35S promoter was frequently silenced after shoot regeneration, whereas maize UBQ promoter achieved stable expression in various young tissues. Binary vectors with Gateway cassettes under the control of the UBQ promoter allowed us to develop stable organelle markers for nuclei, microtubules and P-bodies in rice. The maize UBQ promoter can be easily replaced with any promoters of interest as exemplified by reporters of mitotic cells and provascular bundles. Finally, by introducing two genomic fluorescent reporters, we showed utilities of the Gateway cassette and two selection markers in large DNA fragment transfer and sequential transformations, respectively. Thus, these binary vectors provide useful choices of transgenic experiments in rice.     

Development of transgenic rice plants expressing maize anthocyanin genes and increased blast resistance

The functional association of flavonoids with plant stress responses, though widely reported in the literature, remains to be documented in rice. Towards this end we chose a transgenic approach with well characterized regulatory and structural genes from maize involved in flavonoid biosynthesis. Activation of anthocyanin pathway in rice was investigated with the maize genes. Production of purple anthocyanin pigments were observed in transformed Tp309 (a japonica rice variety) calluses upon the introduction of the maize regulatory genes C1 (coloured-1), R (red) and the structural gene C2 (coloured-2, encoding chalcone synthase). In addition, stable transgenic plants carrying the maize C2 gene under the control of the maize Ubiquitin promoter were generated. A localized appearance of purple/red pigment in the leaf blade and leaf sheath of R₀ C2 transgenic seedlings was observed. Such a patchy pattern of the transgene expression appears to be conditioned by the genetic background of Tp309, which is homozygous for dominant color inhibitor gene(s) whose presence was unravelled by appropriate genetic crosses. Southern blot analysis of the transgenic plants demonstrated that c2 cDNA was integrated into the genome. Western blot analysis of these primary transgenics revealed the CHS protein while it was not detected in the control untransformed Tp3O9, suggesting that Tp309 might have a mutation at the corresponding C2 locus or that the expression of this gene is suppressed in Tp309. Further analysis of C2 transgenics revealed CHS protein only in three out of sixteen plants that were western-positive in the R₀ generation, suggesting gene silencing. Preliminary screening of these R₁ plants against the rice blast fungus Magnaporthe grisea revealed an increase in resistance.     

Dual-origin plasmids containing an amplifiable ColE1 ori; temperature-controlled expression of cloned genes

Dual-origin plasmids comprising an inducible ColE1-derived origin of replication controlled by the λ p_R promoter, the c1857 temperature-sensitive represser gene and the pSC101 origin of replication and its associated par sequence, were constructed. Such plasmids carrying cloned genes were stably maintained at four copies per chromosome, and were readily amplifiable by thermal induction. Cloned gene expression increased with copy number, and accumulation values of > 20% total cellular protein were detected. These vectors should prove useful for the production of foreign protein on a large scale, since they provide for stable plasmid maintenance during the growth phase, and high-level gene expression without plasmid loss during the production phase.