Cisgenic apple trees; development, characterization, and performance

F.A. Krens, J.G. Schaart, A.M. van der Burgh, I.E.M. Tinnenbroek-Capel, R. Groenwold, L.P. Kodde, G.A.L. Broggini, C. Gessler, H.J. Schouten

Research output: Contribution to journalArticleAcademicpeer-review

27 Citations (Scopus)

Abstract

Two methods were developed for the generation of cisgenic apples. Both have been successfully applied producing trees. The first method avoids the use of any foreign selectable marker genes; only the gene-of-interest is integrated between the T-DNA border sequences. The second method makes use of recombinase-based marker excision. For the first method we used the MdMYB10 gene from a red-fleshed apple coding for a transcription factor involved in regulating anthocyanin biosynthesis. Red plantlets were obtained and presence of the cisgene was confirmed. Plantlets were grafted and grown in a greenhouse. After 3 years, the first flowers appeared, showing red petals. Pollination led to production of red-fleshed cisgenic apples. The second method used the pM(arker)F(ree) vector system, introducing the scab resistance gene Rvi6, derived from apple. Agrobacterium-mediated transformation, followed by selection on kanamycin, produced genetically modified apple lines. Next, leaves from in vitro material were treated to activate the recombinase leading to excision of selection genes. Subsequently, the leaf explants were subjected to negative selection for marker-free plantlets by inducing regeneration on medium containing 5-fluorocytosine. After verification of the marker-free nature, the obtained plants were grafted onto rootstocks. Young trees from four cisgenic lines and one intragenic line, all containing Rvi6, were planted in an orchard. Appropriate controls were incorporated in this trial. We scored scab incidence for three consecutive years on leaves after inoculations with Rvi6-avirulent strains. One cisgenic line and the intragenic line performed as well as the resistant control. In 2014 trees started to overcome their juvenile character and formed flowers and fruits. The first results of scoring scab symptoms on apple fruits were obtained. Apple fruits from susceptible controls showed scab symptoms, while fruits from cisgenic and intragenic lines were free of scab.
Original languageEnglish
Article number286
Number of pages11
JournalFrontiers in Plant Science
Volume6
DOIs
Publication statusPublished - 2015

Fingerprint

apples
plantlets
fruits
signs and symptoms (plants)
genes
flowers
leaves
methodology
avirulent strains
kanamycin
Agrobacterium
corolla
rootstocks
anthocyanins
pollination
explants
orchards
transcription factors
biosynthesis
greenhouses

Keywords

  • scab resistance gene
  • selectable marker
  • mediated transformation
  • plant transformation
  • transcription factor
  • transgenic apple
  • agrobacterium
  • gala
  • recombinase
  • expression

Cite this

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title = "Cisgenic apple trees; development, characterization, and performance",
abstract = "Two methods were developed for the generation of cisgenic apples. Both have been successfully applied producing trees. The first method avoids the use of any foreign selectable marker genes; only the gene-of-interest is integrated between the T-DNA border sequences. The second method makes use of recombinase-based marker excision. For the first method we used the MdMYB10 gene from a red-fleshed apple coding for a transcription factor involved in regulating anthocyanin biosynthesis. Red plantlets were obtained and presence of the cisgene was confirmed. Plantlets were grafted and grown in a greenhouse. After 3 years, the first flowers appeared, showing red petals. Pollination led to production of red-fleshed cisgenic apples. The second method used the pM(arker)F(ree) vector system, introducing the scab resistance gene Rvi6, derived from apple. Agrobacterium-mediated transformation, followed by selection on kanamycin, produced genetically modified apple lines. Next, leaves from in vitro material were treated to activate the recombinase leading to excision of selection genes. Subsequently, the leaf explants were subjected to negative selection for marker-free plantlets by inducing regeneration on medium containing 5-fluorocytosine. After verification of the marker-free nature, the obtained plants were grafted onto rootstocks. Young trees from four cisgenic lines and one intragenic line, all containing Rvi6, were planted in an orchard. Appropriate controls were incorporated in this trial. We scored scab incidence for three consecutive years on leaves after inoculations with Rvi6-avirulent strains. One cisgenic line and the intragenic line performed as well as the resistant control. In 2014 trees started to overcome their juvenile character and formed flowers and fruits. The first results of scoring scab symptoms on apple fruits were obtained. Apple fruits from susceptible controls showed scab symptoms, while fruits from cisgenic and intragenic lines were free of scab.",
keywords = "scab resistance gene, selectable marker, mediated transformation, plant transformation, transcription factor, transgenic apple, agrobacterium, gala, recombinase, expression",
author = "F.A. Krens and J.G. Schaart and {van der Burgh}, A.M. and I.E.M. Tinnenbroek-Capel and R. Groenwold and L.P. Kodde and G.A.L. Broggini and C. Gessler and H.J. Schouten",
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doi = "10.3389/fpls.2015.00286",
language = "English",
volume = "6",
journal = "Frontiers in Plant Science",
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Cisgenic apple trees; development, characterization, and performance. / Krens, F.A.; Schaart, J.G.; van der Burgh, A.M.; Tinnenbroek-Capel, I.E.M.; Groenwold, R.; Kodde, L.P.; Broggini, G.A.L.; Gessler, C.; Schouten, H.J.

In: Frontiers in Plant Science, Vol. 6, 286, 2015.

Research output: Contribution to journalArticleAcademicpeer-review

TY - JOUR

T1 - Cisgenic apple trees; development, characterization, and performance

AU - Krens, F.A.

AU - Schaart, J.G.

AU - van der Burgh, A.M.

AU - Tinnenbroek-Capel, I.E.M.

AU - Groenwold, R.

AU - Kodde, L.P.

AU - Broggini, G.A.L.

AU - Gessler, C.

AU - Schouten, H.J.

PY - 2015

Y1 - 2015

N2 - Two methods were developed for the generation of cisgenic apples. Both have been successfully applied producing trees. The first method avoids the use of any foreign selectable marker genes; only the gene-of-interest is integrated between the T-DNA border sequences. The second method makes use of recombinase-based marker excision. For the first method we used the MdMYB10 gene from a red-fleshed apple coding for a transcription factor involved in regulating anthocyanin biosynthesis. Red plantlets were obtained and presence of the cisgene was confirmed. Plantlets were grafted and grown in a greenhouse. After 3 years, the first flowers appeared, showing red petals. Pollination led to production of red-fleshed cisgenic apples. The second method used the pM(arker)F(ree) vector system, introducing the scab resistance gene Rvi6, derived from apple. Agrobacterium-mediated transformation, followed by selection on kanamycin, produced genetically modified apple lines. Next, leaves from in vitro material were treated to activate the recombinase leading to excision of selection genes. Subsequently, the leaf explants were subjected to negative selection for marker-free plantlets by inducing regeneration on medium containing 5-fluorocytosine. After verification of the marker-free nature, the obtained plants were grafted onto rootstocks. Young trees from four cisgenic lines and one intragenic line, all containing Rvi6, were planted in an orchard. Appropriate controls were incorporated in this trial. We scored scab incidence for three consecutive years on leaves after inoculations with Rvi6-avirulent strains. One cisgenic line and the intragenic line performed as well as the resistant control. In 2014 trees started to overcome their juvenile character and formed flowers and fruits. The first results of scoring scab symptoms on apple fruits were obtained. Apple fruits from susceptible controls showed scab symptoms, while fruits from cisgenic and intragenic lines were free of scab.

AB - Two methods were developed for the generation of cisgenic apples. Both have been successfully applied producing trees. The first method avoids the use of any foreign selectable marker genes; only the gene-of-interest is integrated between the T-DNA border sequences. The second method makes use of recombinase-based marker excision. For the first method we used the MdMYB10 gene from a red-fleshed apple coding for a transcription factor involved in regulating anthocyanin biosynthesis. Red plantlets were obtained and presence of the cisgene was confirmed. Plantlets were grafted and grown in a greenhouse. After 3 years, the first flowers appeared, showing red petals. Pollination led to production of red-fleshed cisgenic apples. The second method used the pM(arker)F(ree) vector system, introducing the scab resistance gene Rvi6, derived from apple. Agrobacterium-mediated transformation, followed by selection on kanamycin, produced genetically modified apple lines. Next, leaves from in vitro material were treated to activate the recombinase leading to excision of selection genes. Subsequently, the leaf explants were subjected to negative selection for marker-free plantlets by inducing regeneration on medium containing 5-fluorocytosine. After verification of the marker-free nature, the obtained plants were grafted onto rootstocks. Young trees from four cisgenic lines and one intragenic line, all containing Rvi6, were planted in an orchard. Appropriate controls were incorporated in this trial. We scored scab incidence for three consecutive years on leaves after inoculations with Rvi6-avirulent strains. One cisgenic line and the intragenic line performed as well as the resistant control. In 2014 trees started to overcome their juvenile character and formed flowers and fruits. The first results of scoring scab symptoms on apple fruits were obtained. Apple fruits from susceptible controls showed scab symptoms, while fruits from cisgenic and intragenic lines were free of scab.

KW - scab resistance gene

KW - selectable marker

KW - mediated transformation

KW - plant transformation

KW - transcription factor

KW - transgenic apple

KW - agrobacterium

KW - gala

KW - recombinase

KW - expression

U2 - 10.3389/fpls.2015.00286

DO - 10.3389/fpls.2015.00286

M3 - Article

VL - 6

JO - Frontiers in Plant Science

JF - Frontiers in Plant Science

SN - 1664-462X

M1 - 286

ER -