Protocol:Genetic transformation of the fern ceratopteris richardii through microparticle bombardment

BACKGROUND: The inability to genetically transform any fern species has been a major technical barrier to unlocking fern biology. Initial attempts to overcome this limitation were based on transient transformation approaches or achieved very low efficiencies. A highly efficient method of stable transformation was recently reported using the fern Ceratopteris richardii, in which particle bombardment of callus tissue achieved transformation efficiencies of up to 72%. As such, this transformation method represents a highly desirable research tool for groups wishing to undertake fern genetic analysis. RESULTS: We detail an updated and optimized protocol for transformation of C. richardii by particle bombardment, including all necessary ancillary protocols for successful growth and propagation of this species in a laboratory environment. The C. richardii lifecycle comprises separate, free-living gametophyte and sporophyte stages. Callus is induced from the sporophyte apex through growth on cytokinin-containing tissue culture medium and can be maintained indefinitely by sub-culturing. Transgene DNA is introduced into callus cells through particle bombardment, and stable genomic integration events are selected by regeneration and growth of T(0) sporophytes for a period of 8 weeks on medium containing antibiotics. Selection of T(1) transgenic progeny is accomplished through screening T(1) gametophytes for antibiotic resistance. In many cases sexual reproduction and development of transgenic embryos requires growth and fertilization of gametophytes in the absence of antibiotics, followed by a separate screen for antibiotic resistance in the resultant sporophyte generation. CONCLUSIONS: Genetic transformation of C. richardii using this protocol was found to be robust under a broad range of bombardment and recovery conditions. The successful expansion of the selection toolkit to include a second antibiotic for resistance screening (G-418) and different resistance marker promoters increases the scope of transformations possible using this technique and offers the prospect of more complex analysis, for example the creation of lines carrying more than one transgene. The introduction of a robust and practicable transformation technique is a very important milestone in the field of fern biology, and its successful implementation in C. richardii paves the way for adoption of this species as the first fern genetic model. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13007-015-0080-8) contains supplementary material, which is available to authorized users

LEAFY maintains apical stem cell activity during shoot development in the fern Ceratopteris richardii

During land plant evolution, determinate spore-bearing axes (retained in extant bryophytes such as mosses) were progressively transformed into indeterminate branching shoots with specialized reproductive axes that form flowers. The LEAFY transcription factor, which is required for the first zygotic cell division in mosses and primarily for floral meristem identity in flowering plants, may have facilitated developmental innovations during these transitions. Mapping the LEAFY evolutionary trajectory has been challenging, however, because there is no functional overlap between mosses and flowering plants, and no functional data from intervening lineages. Here, we report a transgenic analysis in the fern Ceratopteris richardii that reveals a role for LEAFY in maintaining cell divisions in the apical stem cells of both haploid and diploid phases of the lifecycle. These results support an evolutionary trajectory in which an ancestral LEAFY module that promotes cell proliferation was progressively co-opted, adapted and specialized as novel shoot developmental contexts emerged

Protocol: genetic transformation of the fern Ceratopteris richardii through microparticle bombardment

BACKGROUND: The inability to genetically transform any fern species has been a major technical barrier to unlocking fern biology. Initial attempts to overcome this limitation were based on transient transformation approaches or achieved very low efficiencies. A highly efficient method of stable transformation was recently reported using the fern Ceratopteris richardii, in which particle bombardment of callus tissue achieved transformation efficiencies of up to 72%. As such, this transformation method represents a highly desirable research tool for groups wishing to undertake fern genetic analysis. RESULTS: We detail an updated and optimized protocol for transformation of C. richardii by particle bombardment, including all necessary ancillary protocols for successful growth and propagation of this species in a laboratory environment. The C. richardii lifecycle comprises separate, free-living gametophyte and sporophyte stages. Callus is induced from the sporophyte apex through growth on cytokinin-containing tissue culture medium and can be maintained indefinitely by sub-culturing. Transgene DNA is introduced into callus cells through particle bombardment, and stable genomic integration events are selected by regeneration and growth of T(0) sporophytes for a period of 8 weeks on medium containing antibiotics. Selection of T(1) transgenic progeny is accomplished through screening T(1) gametophytes for antibiotic resistance. In many cases sexual reproduction and development of transgenic embryos requires growth and fertilization of gametophytes in the absence of antibiotics, followed by a separate screen for antibiotic resistance in the resultant sporophyte generation. CONCLUSIONS: Genetic transformation of C. richardii using this protocol was found to be robust under a broad range of bombardment and recovery conditions. The successful expansion of the selection toolkit to include a second antibiotic for resistance screening (G-418) and different resistance marker promoters increases the scope of transformations possible using this technique and offers the prospect of more complex analysis, for example the creation of lines carrying more than one transgene. The introduction of a robust and practicable transformation technique is a very important milestone in the field of fern biology, and its successful implementation in C. richardii paves the way for adoption of this species as the first fern genetic model. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13007-015-0080-8) contains supplementary material, which is available to authorized users

NIME Array Percentage G + C Content Profile for an Array Located at 1283600–1284640 bp in the FAM18 Genome

<p>The % G + C window size is 24 bases. Maximum, minimum, and median are also shown. Red blocks represent dRS3 with inverted repeats indicated by grey triangles. Blue blocks represent RS elements.</p

Rearrangements between the Meningococcal Genomes

<p>The dotplots were generated using MUMmer version 3.15 (<a href="http://mummer.sourceforge.net" target="_blank">http://mummer.sourceforge.net</a>) and indicate matching sequences with codirectional and reversed regions of synteny shown in red and green, respectively. Genome sequences are aligned to start/finish at the origin of replication with the approximate position of the terminus of replication indicated (Ter) (note this required rotation of the publicly available sequences for Z2491 and MC58, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0030023#s3" target="_blank">Materials and Methods</a>). Also shown are the positions of the foci of the three major inversion events (IE1, IE2, and IE3, see text for detail).</p

Pie Charts Showing Association between Repeat Arrays and Surface Proteins in FAM18

<p>Chart sectors are coloured according to gene function (see colour key).</p

Sequence Divergence in Orthologues Flanking Repeat Arrays<b> </b>

<div><p>(A) Plot of repeat array length against flanking orthologue sequence identity for FAM18 versus Z2491 (blue diamond), Z2491 versus MC58 (red square), and FAM18 versus MC58 (green triangle).</p><p>(B) Plot of distance from array versus orthologue identity for FAM18 versus Z2491, Z2491 versus MC58, and FAM18 versus MC58.</p><p>(C) The same as (B), ignoring the first CDS.</p></div

The DNA sequence of chromosome I of an African trypanosome: gene content, chromosome organisation, recombination and polymorphism

The African trypanosome, Trypanosoma brucei, causes sleeping sickness in humans in sub-Saharan Africa. Here we report the sequence and analysis of the 1.1 Mb chromosome I, which encodes approximately 400 predicted genes organised into directional clusters, of which more than 100 are located in the largest cluster of 250 kb. A 160-kb region consists primarily of three gene families of unknown function, one of which contains a hotspot for retroelement insertion. We also identify five novel gene families. Indeed, almost 20% of predicted genes are members of families. In some cases, tandemly arrayed genes are 99–100% identical, suggesting an active process of amplification and gene conversion. One end of the chromosome consists of a putative bloodstream-form variant surface glycoprotein (VSG) gene expression site that appears truncated and degenerate. The other chromosome end carries VSG and expression site-associated genes and pseudogenes over 50 kb of subtelomeric sequence where, unusually, the telomere-proximal VSG gene is oriented away from the telomere. Our analysis includes the cataloguing of minor genetic variations between the chromosome I homologues and an estimate of crossing-over frequency during genetic exchange. Genetic polymorphisms are exceptionally rare in sequences located within and around the strand-switches between several gene clusters