Development of late blight resistant potatoes by cisgene stacking View Full Text


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Article Info

DATE

2014-05-29

AUTHORS

Kwang-Ryong Jo, Chol-Jun Kim, Sung-Jin Kim, Tok-Yong Kim, Marjan Bergervoet, Maarten A Jongsma, Richard GF Visser, Evert Jacobsen, Jack H Vossen

ABSTRACT

BackgroundPhytophthora infestans, causing late blight in potato, remains one of the most devastating pathogens in potato production and late blight resistance is a top priority in potato breeding. The introduction of multiple resistance (R) genes with different spectra from crossable species into potato varieties is required. Cisgenesis is a promising approach that introduces native genes from the crops own gene pool using GM technology, thereby retaining favourable characteristics of established varieties.ResultsWe pursued a cisgenesis approach to introduce two broad spectrum potato late blight R genes, Rpi-sto1 and Rpi-vnt1.1 from the crossable species Solanum stoloniferum and Solanum venturii, respectively, into three different potato varieties. First, single R gene-containing transgenic plants were produced for all varieties to be used as references for the resistance levels and spectra to be expected in the respective genetic backgrounds. Next, a construct containing both cisgenic late blight R genes (Rpi-vnt1.1 and Rpi-sto1), but lacking the bacterial kanamycin resistance selection marker (NPTII) was transformed to the three selected potato varieties using Agrobacterium-mediated transformation. Gene transfer events were selected by PCR among regenerated shoots. Through further analyses involving morphological evaluations in the greenhouse, responsiveness to Avr genes and late blight resistance in detached leaf assays, the selection was narrowed down to eight independent events. These cisgenic events were selected because they showed broad spectrum late blight resistance due to the activity of both introduced R genes. The marker-free transformation was compared to kanamycin resistance assisted transformation in terms of T-DNA and vector backbone integration frequency. Also, differences in regeneration time and genotype dependency were evaluated.ConclusionsWe developed a marker-free transformation pipeline to select potato plants functionally expressing a stack of late blight R genes. Marker-free transformation is less genotype dependent and less prone to vector backbone integration as compared to marker-assisted transformation. Thereby, this study provides an important tool for the successful deployment of R genes in agriculture and contributes to the production of potentially durable late blight resistant potatoes. More... »

PAGES

50

References to SciGraph publications

  • 2007-06-16. Molecular analysis of Agrobacterium T-DNA integration in tomato reveals a role for left border sequence homology in most integration events in MOLECULAR GENETICS AND GENOMICS
  • 1966-05. New R genes in Solanum demissum lindl. And their complementary races of Phytophthora infestans (Mont.) de bary in EUPHYTICA
  • 2006-01-05. Mapping the R10 and R11 genes for resistance to late blight (Phytophthora infestans) present in the potato (Solanum tuberosum) R-gene differentials of Black in THEORETICAL AND APPLIED GENETICS
  • 2008-03. Cisgenesis, a New Tool for Traditional Plant Breeding, Should be Exempted from the Regulation on Genetically Modified Organisms in a Step by Step Approach in POTATO RESEARCH
  • 1999-04. A Laboratory Assay for Phytophthora infestans Resistance in Various Solanum Species Reflects the Field Situation in EUROPEAN JOURNAL OF PLANT PATHOLOGY
  • 2011-04-10. Functional stacking of three resistance genes against Phytophthora infestans in potato in TRANSGENIC RESEARCH
  • 2013-12-17. A locus conferring effective late blight resistance in potato cultivar Sárpo Mira maps to chromosome XI in THEORETICAL AND APPLIED GENETICS
  • 1991. Regeneration and transformation of potato by Agrobacterium tumefaciens in PLANT TISSUE CULTURE MANUAL
  • 2003-03-10. A transformation method for obtaining marker-free plants of a cross-pollinating and vegetatively propagated crop in NATURE BIOTECHNOLOGY
  • 1995-09. Microprep protocol for extraction of DNA from tomato and other herbaceous plants in PLANT MOLECULAR BIOLOGY REPORTER
  • 2002-03. Transformation of a large number of potato varieties: genotype-dependent variation in efficiency and somaclonal variability in EUPHYTICA
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    PUBMED

    https://www.ncbi.nlm.nih.gov/pubmed/24885731


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    29 schema:description BackgroundPhytophthora infestans, causing late blight in potato, remains one of the most devastating pathogens in potato production and late blight resistance is a top priority in potato breeding. The introduction of multiple resistance (R) genes with different spectra from crossable species into potato varieties is required. Cisgenesis is a promising approach that introduces native genes from the crops own gene pool using GM technology, thereby retaining favourable characteristics of established varieties.ResultsWe pursued a cisgenesis approach to introduce two broad spectrum potato late blight R genes, Rpi-sto1 and Rpi-vnt1.1 from the crossable species Solanum stoloniferum and Solanum venturii, respectively, into three different potato varieties. First, single R gene-containing transgenic plants were produced for all varieties to be used as references for the resistance levels and spectra to be expected in the respective genetic backgrounds. Next, a construct containing both cisgenic late blight R genes (Rpi-vnt1.1 and Rpi-sto1), but lacking the bacterial kanamycin resistance selection marker (NPTII) was transformed to the three selected potato varieties using Agrobacterium-mediated transformation. Gene transfer events were selected by PCR among regenerated shoots. Through further analyses involving morphological evaluations in the greenhouse, responsiveness to Avr genes and late blight resistance in detached leaf assays, the selection was narrowed down to eight independent events. These cisgenic events were selected because they showed broad spectrum late blight resistance due to the activity of both introduced R genes. The marker-free transformation was compared to kanamycin resistance assisted transformation in terms of T-DNA and vector backbone integration frequency. Also, differences in regeneration time and genotype dependency were evaluated.ConclusionsWe developed a marker-free transformation pipeline to select potato plants functionally expressing a stack of late blight R genes. Marker-free transformation is less genotype dependent and less prone to vector backbone integration as compared to marker-assisted transformation. Thereby, this study provides an important tool for the successful deployment of R genes in agriculture and contributes to the production of potentially durable late blight resistant potatoes.
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    36 schema:keywords Agrobacterium
    37 Avr genes
    38 ConclusionsWe
    39 Further analysis
    40 GM technology
    41 PCR
    42 R genes
    43 ResultsWe
    44 Rpi-sto1
    45 Rpi-vnt1.1
    46 Solanum stoloniferum
    47 Solanum venturii
    48 T-DNA
    49 activity
    50 agriculture
    51 analysis
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    53 assays
    54 background
    55 blight
    56 blight resistance
    57 blight resistant potato
    58 breeding
    59 broad-spectrum late blight resistance
    60 characteristics
    61 cisgenes
    62 cisgenesis
    63 constructs
    64 crops
    65 crossable species
    66 dependency
    67 deployment
    68 detached leaf assays
    69 devastating pathogen
    70 development
    71 differences
    72 different potato varieties
    73 different spectra
    74 evaluation
    75 events
    76 favorable characteristics
    77 frequency
    78 gene pool
    79 gene transfer events
    80 genes
    81 genetic background
    82 genotype dependency
    83 genotypes
    84 greenhouse
    85 important tool
    86 independent events
    87 infestans
    88 integration
    89 integration frequency
    90 introduction
    91 kanamycin resistance
    92 late blight
    93 late blight R genes
    94 late blight resistance
    95 late blight resistant potato
    96 leaf assays
    97 less genotype
    98 levels
    99 marker-free transformation
    100 markers
    101 morphological evaluation
    102 multiple resistance genes
    103 native genes
    104 pathogens
    105 pipeline
    106 plants
    107 pool
    108 potato
    109 potato breeding
    110 potato plants
    111 potato production
    112 potato varieties
    113 priority
    114 production
    115 promising approach
    116 reference
    117 regenerated shoots
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    119 resistance
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    121 resistance levels
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    123 respective genetic backgrounds
    124 responsiveness
    125 selection
    126 selection marker
    127 shoots
    128 species
    129 spectra
    130 stack
    131 stoloniferum
    132 study
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    134 technology
    135 terms
    136 time
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