ultivated peanut or groundnut (A. hypogaea L.) is among the most important oil and food legumes, grown on 25 million ha between latitudes 40° N and 40° S with annual production of ~46 million tons (http://www.fao.org/faostat/en/#home). It presumably was domesticated in South America ~6,000 years ago and then was widely distributed in post-Columbian times 1. Combining richness in seed oil (~46-58%) and protein (~22-32%), peanut is important in fighting malnutrition and ensuring food security.

Direct repeats ofkaryotyping ͉ transformation ͉ chromosome engineering

Engineered minichromosomes were constructed in maize by modifying natural A and supernumerary B chromosomes. By using telomere-mediated chromosomal truncation, it was demonstrated that such an approach is feasible for the generation of minichromosomes of normal A chromosomes by selection of spontaneous polyploid events that compensate for the deficiencies produced. B chromosomes are readily fractionated by biolistic transformation of truncating plasmids. Foreign genes were faithfully expressed from integrations into normal B chromosomes and from truncated miniB chromosomes. Site-specific recombination between the terminal transgene on a miniA chromosome and a terminal site on a normal chromosome was demonstrated. It was also found that the miniA chromosome did not pair with its progenitor chromosomes during meiosis, indicating a useful property for such constructs. The miniB chromosomes are faithfully transmitted from one generation to the next but can be changed in dosage in the presence of normal B chromosomes. This approach for construction of engineered chromosomes can be easily extended to other plant species because it does not rely on cloned centromere sequences, which are species-specific. These platforms will provide avenues for studies on plant chromosome structure and function and for future developments in biotechnology and agriculture.artificial chromosomes ͉ FISH ͉ genetic engineering ͉ telomere truncation A rtificial chromosomes involving de novo centromere formation on an independently assembled unit and engineered minichromosomes produced by telomere truncation provide striking advantages over traditional methods of gene transformation in yeast and mammalian cells (1-5). The development of such chromosomes in plants would provide these advantages for many applications in basic studies, biotechnology, and agriculture. These chromosomes could be used as independent platforms for foreign gene expression without random integration into the normal chromosomes. Further additions of unlimited amounts of DNA could be added to these platforms in a sequential manner via different site-specific recombination cassettes. Genes introduced in this way would be present in a defined context and thus could be expressed at a more predictable level than through random integration (6). Hence, additional genes, multigene complexes, or even whole metabolic pathways could potentially be added to a genotype. Moreover, engineered or artificial chromosomes could be easily introduced or removed from a genotype by genetic crosses and would facilitate introgression of transgenes to different genetic backgrounds.To extend engineered chromosome technology to plants, we developed a method of telomere-mediated chromosomal truncation in maize by Agrobacterium-mediated transformation of constructs with multiple copies of the telomere sequence (7). Here, we report the use of this technology to produce minichromosome platforms by truncating both normal A and supernumerary B maize chromosomes and at the same time introducing site-spe...

The gene VI product (P6) of Cauliflower mosaic virus (CaMV) is a multifunctional protein known to be a major component of cytoplasmic inclusion bodies formed during CaMV infection. Although these inclusions are known to contain virions and are thought to be sites of translation from the CaMV 35S polycistronic RNA intermediate, the precise role of these bodies in the CaMV infection cycle remains unclear. Here, we examine the functionality and intracellular location of a fusion between P6 and GFP (P6-GFP). We initially show that the ability of P6-GFP to transactivate translation is comparable to unmodified P6. Consequently, our work has direct application for the large body of literature in which P6 has been expressed ectopically and its functions characterized. We subsequently found that P6-GFP forms highly motile cytoplasmic inclusion bodies and revealed through fluorescence colocalization studies that these P6-GFP bodies associate with the actin/endoplasmic reticulum network as well as microtubules. We demonstrate that while P6-GFP inclusions traffic along microfilaments, those associated with microtubules appear stationary. Additionally, inhibitor studies reveal that the intracellular movement of P6-GFP inclusions is sensitive to the actin inhibitor, latrunculin B, which also inhibits the formation of local lesions by CaMV in Nicotiana edwardsonii leaves. The motility of P6 along microfilaments represents an entirely new property for this protein, and these results imply a role for P6 in intracellular and cell-to-cell movement of CaMV.

High-frequency transformation of maize (Zea mays L.) using standard binary vectors is advantageous for functional genomics and other genetic engineering studies. Recent advances in Agrobacterium tumefaciens-mediated transformation of maize have made it possible for the public to transform maize using standard binary vectors without a need of the superbinary vector. While maize Hi-II has been a preferred maize genotype to use in various maize transformation efforts, there is still potential and need in further improving its transformation frequency. Here we report the enhanced Agrobacterium-mediated transformation of immature zygotic embryos of maize Hi-II using standard binary vectors. This improved transformation process employs low-salt media in combined use with antioxidant L-cysteine alone or L-cysteine and dithiothreitol (DTT) during the Agrobacterium infection stage. Three levels of N6 medium salts, 10, 50, and 100%, were tested. Both 10 and 50% salts were found to enhance the T-DNA transfer in Hi-II. Addition of DTT to the cocultivation medium also improves the T-DNA transformation. About 12% overall and the highest average of 18% transformation frequencies were achieved from a large number of experiments using immature embryos grown in various seasons. The enhanced transformation protocol established here will be advantageous for maize genetic engineering studies including transformation-based functional genomics.

With the advent of engineered minichromosome technology in plants, an understanding of the properties of small chromosomes is desirable. Twenty-two minichromosomes of related origin but varying in size are described that provide a unique resource to study such behavior. Fourteen minichromosomes from this set could pair with each other in meiotic prophase at frequencies between 25 and 100%, but for the smaller chromosomes, the sister chromatids precociously separated in anaphase I. The other eight minichromosomes did not pair with themselves, and the sister chromatids divided equationally at meiosis I. In plants containing one minichromosome, the sister chromatids also separated at meiosis I. In anaphase II, the minichromosomes progressed to one pole or the other. The maize (Zea mays) Shugoshin protein, which has been hypothesized to protect centromere cohesion in meiosis I, is still present at anaphase I on minichromosomes that divide equationally. Also, there were no differences in the level of phosphorylation of Ser-10 of histone H3, a correlate of cohesion, in the minichromosomes in which sister chromatids separated during anaphase I compared with the normal chromosomes. These analyses suggest that meiotic centromeric cohesion is compromised in minichromosomes depending on their size and cannot be maintained by the mechanisms used by normal-sized chromosomes.

Supernumerary or B chromosomes are selfish entities that maintain themselves in populations by accumulation mechanisms. The accumulation mechanism of the B chromosome of maize (Zea mays) involves nondisjunction at the second pollen mitosis, placing two copies of the B chromosome into one of the two sperm. The B chromosome long arm must be present in the same nucleus for the centromere to undergo nondisjunction. A centromere, containing all of the normal DNA elements, translocated from the B chromosome to the short arm of chromosome 9 was recently found to be epigenetically silenced for centromeric function. When intact B chromosomes were added to this genotype, thus supplying the long arm, the inactive centromere regained the property of nondisjunction causing the translocation chromosome 9 to be differentially distributed to the two sperm or resulted in chromosome breaks in 9S, occasionally producing new translocations. Translocation of the inactive B centromere to chromosome 7 transferred the nondisjunction property to this chromosome. The results provide insight into the molecular and evolutionary basis of this B chromosome accumulation mechanism by demonstrating that nondisjunction is caused by a process that does not depend on normal centromere function but that the region of the chromosome required for nondisjunction resides in the centromeric region.

SUMMARYTelomere truncation has been shown to be an efficient technology for the creation of mini-chromosomes that can be used as artificial chromosome platforms for genetic engineering. Artificial chromosome-based genetic engineering is considered to be superior to the existing techniques of randomized gene integration by Agrobacterium or biolistic-mediated genetic transformation. It organizes multiple transgenes as a unique genetic linkage block for subsequent manipulations in breeding. Telomere truncation technology relies on three components: the telomere sequence that mediates chromosomal truncation, a selection marker that allows the selection of transgenic events, and a site-specific recombination system that can be used to accept future genes into the mini-chromosome by gene targeting. These elements are usually pre-assembled before transformation, a process that is both time and labor consuming. We found in this research that the three elements could be mixed to transform plant cells in a biolistic transformation, and produced efficient chromosomal truncations and mini-chromosomes in rice. This system will allow rapid construction of minichromosomes with a flexible selection of resistant markers, site-specific recombination systems and other desirable elements. In addition, a rice telotrisomic line was used as the starting material for chromosomal truncations. Mini-chromosomes from the truncations of both the telocentric chromosome and other chromosomes were recovered. The mini-chromosomes remained stable during 2 years of subculture. The construction of mini-chromosomes in rice, an economically important crop, will provide a platform for future artificial chromosome-based genetic engineering of rice for stacking multiple genes.