Genetic transformation is a powerful means for the improvement of crop plants, but requires labor‐ and resource‐intensive methods. An efficient method for identifying single‐copy transgene insertion events from a population of independent transgenic lines is desirable. Currently, transgene copy number is estimated by either Southern blot hybridization analyses or quantitative polymerase chain reaction (
RepB C-terminus mutation of a pRi-repABC binary vector affects plasmid copy number in Agrobacterium and transgene copy number in plants
A native repABC replication origin from pRiA4b was previously reported as a single copy plasmid in Agrobacterium tumefaciens and can improve the production of transgenic plants with a single copy insertion of transgenes when it is used in binary vectors for Agrobacter-ium-mediated transformation. A high copy pRi-repABC variant plasmid, pTF::Ri, which does not improve the frequency of single copy transgenic plants, has been reported in the litera-ture. Sequencing the high copy pTF::Ri repABC operon revealed the presence of two muta-tions: one silent mutation and one missense mutation that changes a tyrosine to a histidine (Y299H) in a highly conserved area of the C-terminus of the RepB protein (RepBY299H). Reproducing these mutations in the wild-type pRi-repABC binary vector showed that Agro-bacterium cells with the RepBY299H mutation grow faster on both solidified and in liquid medium, and have higher plasmid copy number as determined by ddPCR. In order to inves-tigate the impact of the RepBY299H mutation on transformation and quality plant production, the RepBY299H mutated pRi-repABC binary vector was compared with the original wild-type pRi-repABC binary vector and a multi-copy oriV binary vector in canola transformation. Molecular analyses of the canola transgenic plants demonstrated that the multi-copy pRi-repABC with the RepBY299H mutation provides no advantage in generating high frequency single copy, backbone-free transgenic plants in comparison with the single copy wild-type pRi-repABC binary vector.
more »
« less
- Award ID(s):
- 1725122
- NSF-PAR ID:
- 10161874
- Date Published:
- Journal Name:
- PloS one
- Volume:
- 13
- Issue:
- 11
- ISSN:
- 1932-6203
- Page Range / eLocation ID:
- e0200972
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
more » « less
Summary qPCR ) experiments. Southern hybridization is a convincing and reliable method, but it also is expensive, time‐consuming and often requires a large amount of genomicDNA and radioactively labeled probes. Alternatively,qPCR requires lessDNA and is potentially simpler to perform, but its results can lack the accuracy and precision needed to confidently distinguish between one‐ and two‐copy events in transgenic plants with large genomes. To address this need, we developed a droplet digitalPCR ‐based method for transgene copy number measurement in an array of crops: rice, citrus, potato, maize, tomato and wheat. The method utilizes specific primers to amplify target transgenes, and endogenous reference genes in a single duplexed reaction containing thousands of droplets. Endpoint amplicon production in the droplets is detected and quantified using sequence‐specific fluorescently labeled probes. The results demonstrate that this approach can generate confident copy number measurements in independent transgenic lines in these crop species. This method and the compendium of probes and primers will be a useful resource for the plant research community, enabling the simple and accurate determination of transgene copy number in these six important crop species. -
For maize genome-editing and bioengineering, genetic transformation of inbred genotypes is most desired due to the uniformity of genetic background in their progenies. However, most maize inbred lines are recalcitrant to tissue culture and transformation. A public, transformable maize inbred B104 has been widely used for genome editing in recent years. This is primarily due to its high degree of genetic similarity shared with B73, an inbred of the reference genome and parent of many breeding populations. Conventional B104 maize transformation protocol requires 16–22 weeks to produce rooted transgenic plants with an average of 4% transformation frequency (number of T0 plants per 100 infected embryos). In this Method paper, we describe an advanced B104 transformation protocol that requires only 7–10 weeks to generate transgenic plants with an average of 6.4% transformation frequency. Over 66% of transgenic plants carried CRISPR/Cas9-induced indel mutations on the target gene, demonstrating that this protocol can be used for genome editing applications. Following the detailed and stepwise procedure described here, this quick and simplified method using the Agrobacterium ternary vector system consisting of a T-DNA binary vector and a compatible helper plasmid can be readily transferable to interested researchers.more » « less
-
more » « less
Abstract Copy number variation (
CNV ) is a major part of the genetic diversity segregating within populations, but remains poorly understood relative to single nucleotide variation. Here, we report on atRNA ligase gene (Migut.N02091;RLG 1a) exhibiting unprecedented, and fitness‐relevant,CNV within an annual population of the yellow monkeyflowerMimulus guttatus .RLG 1a variation was associated with multiple traits in pooled population sequencing (PoolSeq) scans of phenotypic and phenological cohorts. Resequencing of inbred lines revealed intermediate‐frequency three‐copy variants ofRLG 1a (trip+; 5/35 = 14%), andtrip+ lines exhibited elevatedRLG 1a expression under multiple conditions.trip+ carriers, in addition to being over‐represented in late‐flowering and large‐flowered PoolSeq populations, flowered later under stressful conditions in a greenhouse experiment (p < 0.05). In wild population samples, we discovered an additional rareRLG 1a variant (high+ ) that carries 250–300 copies ofRLG 1a totalling ~5.7 Mb (20–40% of a chromosome). In the progeny of ahigh+ carrier, Mendelian segregation of diagnostic alleles andqPCR ‐based copy counts indicate thathigh+ is a single tandem array unlinked to the single‐copyRLG 1a locus. In the wild,high+ carriers had highest fitness in two particularly dry and/or hot years (2015 and 2017; bothp < 0.01), while single‐copy individuals were twice as fecund as eitherCNV type in a lush year (2016:p < 0.005). Our results demonstrate fluctuating selection onCNV s affecting phenological traits in a wild population, suggest that planttRNA ligases mediate stress‐responsive life‐history traits, and introduce a novel system for investigating the molecular mechanisms of gene amplification. -
Efficient genetic transformation is a prerequisite for rapid gene functional analyses and crop trait improvements. We recently demonstrated that new T-DNA binary vectors with NptII/G418 selection and a compatible helper plasmid can efficiently transform maize inbred B104 using our rapid Agrobacterium-mediated transformation method. In this work, we implemented the non-integrating Wuschel2 (Wus2) T-DNA vector method for Agrobacterium-mediated B104 transformation and tested its potential for recalcitrant inbred B73 transformation and gene editing. The non-integrating Wus2 (NIW) T-DNA vector-assisted transformation method uses two Agrobacterium strains: one carrying a gene-of-interest (GOI) construct and the other providing an NIW construct. To monitor Wus2 co-integration into the maize genome, we combined the maize Wus2 expression cassette driven by a strong constitutive promoter with a new visible marker RUBY, which produces the purple pigment betalain. As a GOI construct, we used a previously tested CRISPR-Cas9 construct pKL2359 for Glossy2 gene mutagenesis. When both GOI and NIW constructs were delivered by LBA4404Thy- strain, B104 transformation frequency was significantly enhanced by about two-fold (10% vs. 18.8%). Importantly, we were able to transform a recalcitrant inbred B73 using the NIW-assisted transformation method and obtained three transgene-free edited plants by omitting the selection agent G418. These results suggest that NIW-assisted transformation can improve maize B104 transformation frequency and provide a novel option for CRISPR technology for transgene-free genome editing.more » « less
-
null (Ed.)It has been challenging to simultaneously improve photosynthesis and stress tolerance in plants. Crassulacean acid metabolism (CAM) is a CO2-concentrating mechanism that facilitates plant adaptation to water-limited environments. We hypothesized that the ectopic expression of a CAM-specific phosphoenolpyruvate carboxylase (PEPC), an enzyme that catalyzes primary CO2 fixation in CAM plants, would enhance both photosynthesis and abiotic stress tolerance. To test this hypothesis, we engineered a CAM-specific PEPC gene (named AaPEPC1) from Agave americana into tobacco. In comparison with wild-type and empty vector controls, transgenic tobacco plants constitutively expressing AaPEPC1 showed a higher photosynthetic rate and biomass production under normal conditions, along with significant carbon metabolism changes in malate accumulation, the carbon isotope ratio δ13C, and the expression of multiple orthologs of CAM-related genes. Furthermore, AaPEPC1 overexpression enhanced proline biosynthesis, and improved salt and drought tolerance in the transgenic plants. Under salt and drought stress conditions, the dry weight of transgenic tobacco plants overexpressing AaPEPC1 was increased by up to 81.8% and 37.2%, respectively, in comparison with wild-type plants. Our findings open a new door to the simultaneous improvement of photosynthesis and stress tolerance in plants.more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1371/journal.pone.0200972
