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

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Protocol : Genetic transformation of the fern ceratopteris richardii through microparticle bombardment. / Plackett, Andrew R.G.; Rabbinowitsch, Ester H.; Langdale, Jane A.

In: Plant Methods, Vol. 11, No. 1, 37, 03.07.2015.

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@article{b9eaed4ddf9d4cab82f347b7edc3e788,
title = "Protocol: Genetic transformation of the fern ceratopteris richardii through microparticle bombardment",
abstract = "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 T0 sporophytes for a period of 8weeks on medium containing antibiotics. Selection of T1 transgenic progeny is accomplished through screening T1 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.",
keywords = "Ceratopteris, Fern, Microparticle, Protocol, Transformation, Transgenic",
author = "Plackett, {Andrew R.G.} and Rabbinowitsch, {Ester H.} and Langdale, {Jane A.}",
year = "2015",
month = jul
day = "3",
doi = "10.1186/s13007-015-0080-8",
language = "English",
volume = "11",
journal = "Plant Methods",
issn = "1746-4811",
publisher = "BioMed Central Ltd",
number = "1",

}

RIS

TY - JOUR

T1 - Protocol

T2 - Genetic transformation of the fern ceratopteris richardii through microparticle bombardment

AU - Plackett, Andrew R.G.

AU - Rabbinowitsch, Ester H.

AU - Langdale, Jane A.

PY - 2015/7/3

Y1 - 2015/7/3

N2 - 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 T0 sporophytes for a period of 8weeks on medium containing antibiotics. Selection of T1 transgenic progeny is accomplished through screening T1 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.

AB - 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 T0 sporophytes for a period of 8weeks on medium containing antibiotics. Selection of T1 transgenic progeny is accomplished through screening T1 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.

KW - Ceratopteris

KW - Fern

KW - Microparticle

KW - Protocol

KW - Transformation

KW - Transgenic

UR - http://www.scopus.com/inward/record.url?scp=85027938774&partnerID=8YFLogxK

U2 - 10.1186/s13007-015-0080-8

DO - 10.1186/s13007-015-0080-8

M3 - Article

AN - SCOPUS:85027938774

VL - 11

JO - Plant Methods

JF - Plant Methods

SN - 1746-4811

IS - 1

M1 - 37

ER -