Meiotic Transmission of an In Vitro–Assembled Autonomous Maize Minichromosome

Abstract

Autonomous chromosomes are generated in yeast (yeast artificial chromosomes) and human fibrosarcoma cells (human artificial chromosomes) by introducing purified DNA fragments that nucleate a kinetochore, replicate, and segregate to daughter cells. These autonomous minichromosomes are convenient for manipulating and delivering DNA segments containing multiple genes. In contrast, commercial production of transgenic crops relies on methods that integrate one or a few genes into host chromosomes; extensive screening to identify insertions with the desired expression level, copy number, structure, and genomic location; and long breeding programs to produce varieties that carry multiple transgenes. As a step toward improving transgenic crop production, we report the development of autonomous maize minichromosomes (MMCs). We constructed circular MMCs by combining DsRed and nptII marker genes with 7–190 kb of genomic maize DNA fragments containing satellites, retroelements, and/or other repeats commonly found in centromeres and using particle bombardment to deliver these constructs into embryogenic maize tissue. We selected transformed cells, regenerated plants, and propagated their progeny for multiple generations in the absence of selection. Fluorescent in situ hybridization and segregation analysis demonstrated that autonomous MMCs can be mitotically and meiotically maintained. The MMC described here showed meiotic segregation ratios approaching Mendelian inheritance: 93% transmission as a disome (100% expected), 39% transmission as a monosome crossed to wild type (50% expected), and 59% transmission in self crosses (75% expected). The fluorescent DsRed reporter gene on the MMC was expressed through four generations, and Southern blot analysis indicated the encoded genes were intact. This novel approach for plant transformation can facilitate crop biotechnology by (i) combining several trait genes on a single DNA fragment, (ii) arranging genes in a defined sequence context for more consistent gene expression, and (iii) providing an independent linkage group that can be rapidly introgressed into various germplasms.

Functional analysis of theArabidopsiscentromere by T-DNA insertion-induced centromere breakage

“…Similarly, a ring minichromosome containing human alphoid sequences was shown to be transmissible to the offspring through mouse spermatogenesis (32). Recently, meiotic transmission of circular centromeric DNA, which was introduced into embryonic tissues, was reported for maize, although neither circularity nor kinetochore formation has been proved (33). Nevertheless, our studies indicate that small ring-shaped chromosomes might be useful as chromosomal vectors.…”

Molecular Plant Breeding as the Foundation for 21st Century Crop Improvement

“…yieldgardvt.com/VTScience/Default.aspx), or the combination of transgenes that simultaneously increase synthesis and decrease catabolism of Lys in maize seeds (Frizzi et al, 2008). Recent reports of improvements in gene targeting technology (Ow, 2007) and the construction of meiotically transmissible plant minichromosomes (Carlson et al, 2007;Yu et al, 2007) pave the way for introducing more traits with increasing complexity. With such advances, biotechnology is now poised to assemble useful genetic diversity from essentially any source into constructs that concentrate favorable gene action and maximize heritability for a greatly expanded set of traits.…”

Bioactive Beads-Mediated Transformation of Rice with Large DNA Fragments Containing Aegilops tauschii Genes, with Special Reference to Bead-Production Methodology

“…However, there is no such system for plants. Recently three reports have been published on the production of plant artificial chromosomes (Carlson et al, 2007;Yu et al, 2007;Ananiev et al, 2009). However, there is still no report of utilizing them for plant transformation with large DNA fragments.…”