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United States Department of Agriculture

Agricultural Research Service

Research Project: GENETIC IMPROVEMENT OF CITRUS Title: Transgenic expression in citrus of Vitis MybA1 from a bidirectional promoter resulted in variable anthocyanin expression and was not suitable as a screenable marker without antibiotic selection

Authors
item Stover, Ed
item Avila, Yolanda -
item Li, Zhijian -
item Gray, Dennis -

Submitted to: Journal of Florida State Horticulture Society
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: September 16, 2013
Publication Date: May 1, 2014
Citation: Stover, E., Avila, Y., Li, Z.T., Gray, D. Transgenic expression in citrus of Vitis MybA1 from a bidirectional promoter resulted in variable anthocyanin expression and was not suitable as a screenable marker without antibiotic selection. In: Proceedings of the Florida State Horticulture Science. 126:84-88.

Interpretive Summary: Biotechnology offers potential solutions for major problems facing Florida citrus. Consumer concerns about biotech crops may be reduced if only genes from the same crop species are used. In existing biotech crops, antibiotic resistance genes are included in combination with the desired gene. When new shoots form during exposure to the antibiotic (included in the Petri plate medium) mainly shoots with the new genes are produced, since shoots without the resistance gene are suppressed. This project tested a grape gene as a visual marker, producing purple pigment in shoots with introduced genes. Biotech shoot recovery, with and without inclusion of the antibiotic, was studied using ‘Hamlin’ and Carrizo citrus types. Over sixteen separate experiments 3300 shoot pieces of Carrizo and ‘Hamlin’ were treated. In all cases, 6-20 fold more shoots resulted when antibiotic was not used, since many shoots without new genes were formed. However, no shoots with red pigmentation were recovered when antibiotic was excluded. When antibiotic was included, 28% of resulting ‘Hamlin’ shoots (6 out of 21) and 57% of Carrizo shoots (321/405) had purple color showing that new genes were present. Several deep red Carrizo shoots have grown well in the greenhouse and will be helpful as research tools and may have value as ornamental plants.

Technical Abstract: Transgenic strategies offer potential solutions for major problems facing Florida citrus. Intragenics, in which all transgene components are from the target species’ gene pool, may alleviate consumer concerns and might also facilitate deregulation. Resistance to antibiotics is typically used as a selectable marker for plant transformation and this project was initiated to test a plant gene as a visual marker and an intragenic alternative. Transformation with Vitis MybA1 has been demonstrated to produce purple shoots in grape and was tested as a proof of concept for use of plant-pigmentation markers in citrus transformation. ‘Hamlin’ and Carrizo epicotyls segments were exposed to A. tumefasciens EHA105 containing the DAT or DEAT vector with Vitis MybA1driven by the D35S promoter and neomycin phosphotransferase II (NptII, conferring kanamycin resistance, originally isolated from E. coli), was driven by the Nos promoter. Transformation was compared with and without kanamycin (100 mg/L) in the shoot regeneration medium. In sum over sixteen separate experiments >1300 explants each of Carrizo and ‘Hamlin’ were treated. In all cases, 6-20 fold more shoots resulted when kanamycin was excluded from the medium, since there was no negative selection against non-transformed shoots, but no shoots with red pigmentation were recovered. When kanamycin was included, 28% of resulting ‘Hamlin’ shoots (6 out of 21) and 57% of Carrizo shoots (321/405) displayed anthocyanins. Phenotypes recovered included plants with blotchy reddish leaves and plants with cupped leaves. Pigmented ‘Hamlin’ shoots were very weak and subsequently died. Several deep red Carrizo transformants resulted with potential as research tools and ornamentals.

Last Modified: 10/21/2014
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