Particle bombardment of truncating constructs and methods:
Transgene-induced truncation of endogenous chromosomes can be achieved with particle bombardment. Embryogenic callus and immature embryos have both been tested and successfully transformed with truncating constructs (Yu et al., 2007; Gaeta et al., 2011) using standard particle bombardment methods described by Frame et al., 2000. Constructs containing telomere repeat sequences (400 bp-2.8 kb) have been bombarded as circular plasmids (Yu et al., 2007; Gaeta et al., 2011) or linear fragments (transgene cassette containing 400 bp of telomere repeats at its 3’ end was digested and purified from its vector; R. Gaeta unpublished data). Telomere repeats of 0.5-2.8 kb (PCR amplified or digested from plasmids) have also been cobombarded with linear transgene cassettes but lacking telomere (R. Gaeta and L. Krishnaswamy, unpublished data). Telomere-mediated truncation of endogenous chromosomes has been achieved in every case. For cobombardment, a 1:1 molar ratio of transgene cassette DNA and telomere DNA is prepared and precipitated onto gold. Successful transformation has been achieved using 25 ng-100 ng of selection gene cassette per shot (one plate of ~40 embryos or 40 embryogenic callus pieces; see Frame et al., 2000). Control experiments have been performed using cassette DNA alone or plasmids lacking telomere repeats, and no events have been found to contain truncations or translocations (Yu et al., 2007; R. Gaeta and L. Krishnaswamy unpublished).
Agrobacterium-mediated truncation constructs and methods:
Telomere truncation has also been achieved using Agrobacterium mediated transformation of immature maize embryos with telomere truncation T-DNA constructs (Yu et al., 2006). In these constructs, telomere repeat DNA (up to 2.8 kb) was included at the 3’ of the transgene cassette inside the right border. As with particle bombardment, transgene cassettes containing telomere repeats cause chromosome truncation upon integration. Currently we use the methods described in Vega et al., 2008 for Agrobacterium infection.
General overview of minichromosome production and detection:
Chromosomes truncations have repeatedly been produced from both A and B chromosomes in maize following transformation with telomere containing DNAs (Yu et al., 2006; 2007; Gaeta et al., 2011; R. Gaeta and L. Krishnaswamy unpublished data). Fluorescence in situ hybridization (FISH) is the most effective method for identifying chromosome truncations, and transgenes are often detected at or near the site of truncation; however, the technique is limited to the detection of larger truncations that are visibly different from the intact homologous chromosome. Small truncations can be detected or verified by Southern blot using a transgene probe adjacent to the telomere sequences in the truncating construct (See Yu et al., 2006). Primer extension telomere repeat amplification (PETRA) has also been used to detect telomere truncation events in Arabidopsis (Nelson et al., 2011). In some cases FISH experiments have detected transgenes at the terminus of a truncated chromosome, but the terminal location could not be verified by southern blot. This was observed in an experiment in which a circular plasmid containing a telomere sequence was bombarded (Gaeta et al., 2011). In a cobombardment experiment, a truncated chromosome was identified which had an interstitial transgene locus on the truncated arm (R. Gaeta, unpublished). Very small A and B-chromosome derived truncations (minichromosomes) can be distinguished using B-repeat and centromere-specific probes (Masonbrink et al., 2010). A-chromosome truncations exhibit ovule abortion on the ears, which have uneven seed rows, while B-chromosome truncations generally show no phenotype. As with other transgenes containing the Bar selection gene for transformation, plants containing minichromosomes will express resistance to Bialophos and their transmission can be followed by this phenotype.
Karyotyping & FISH Manual: Maize Karyotyping_&_FISH_Manual_2015.pdf
- Gaeta RT, Danilova TV, Zhao C, Masonbrink RE, McCaw ME, Birchler JA. 2011 Recovery of a telomere-truncated chromosome via a compensating translocation in maize. Genome 54: 184-195.
- Masonbrink RM and Birchler JA 2010. Sporophytic nondisjunctionof the maize B chromosome at high copy numbers. J. Genet. Genomics, 37(1): 79–84. doi:10.1016/S1673-8527(09)60027-8.
- Nelson AD, Lamb JC, Kobrossly PS, and Shippen DE. 2011 Parameters affecting telomere-mediated chromosomal truncation in Arabidopsis. Plant Cell doi: 10.1105/tpc.111.086017.
- Yu, W., Lamb, J.C., Han, F., and Birchler, J.A. 2006. Telomere mediated chromosomal truncation in maize. Proc. Natl. Acad.Sci. U.S.A. 103(46): 17331–17336. doi:10.1073/pnas.0605750103. PMID:17085598.
- Yu, W., Han, F., Gao, Z., Vega, J.M., and Birchler, J.A. 2007.Construction and behavior of engineered minichromosomes in maize. Proc. Natl. Acad. Sci. U.S.A. 104(21): 8924–8929.doi:10.1073/pnas.0700932104. PMID:17502617.