Selectable marker-free transgenic orange plants recovered under non-selective conditions and through PCR analysis of all regenerants

Abstract

Selectable marker (SM) genes have been considered necessary to achieve acceptable rates in the generation of transgenic plants. Genes encoding antibiotic or herbicide resistance are widely used for this purpose. In most cases, once transgenic plants have been regenerated, permanence of SM genes in the plant genome is no longer necessary, and it becomes a matter of public concern. Moreover, the removal of SM genes from transgenic plants could facilitate gene stacking through successive transformations, particularly when the availability of these markers is rather limited for most crop plants. In the genus Citrus, with highly heterozygotic species of long generation cycles, methods implying the segregation and removal of marker transgenes in the progeny are not feasible. Here, we have evaluated the direct production of SM-free citrus plants under non-selective conditions, using a ''clean'' binary vector carrying only the transgene of interest, and through the recovery of transformants by polymerase chain reaction (PCR) analysis of all regenerated shoots. The response of two different citrus genotypes, Carrizo citrange (intergeneric hybrid of C. sinensis L. Osb. X Poncirus trifoliata L. Raf.) and Pineapple sweet orange (C. sinensis L. Osb.), was evaluated. Our results indicate that, in this system, the competence between transgenic and nontransgenic cells is the main factor determining final transgenic regeneration frequencies. For Carrizo citrange, no transgenic plant could be recovered. For Pineapple sweet orange, marker-free transformation efficiency was 1.7%, paving the way for the viable production of orange transformants carrying only the transgene(s) of interest.

An Efficient Intragenic Vector for Generating Intragenic and Cisgenic Plants in Citrus

“…Similar levels of transformation efficiency were observed in the production of intra-/cisgenic apple plants [13]. Based on our current transformation protocol, the efficiency is acceptable for generating "foreign DNA-free" intra-/cisgenic plants in citrus [7,14,15].…”

“…In citrus, although there is a report on recovering selectable marker-free transgenic orange plant under non-selective conditions, no intra-/cisgenesis experiment has been conducted due to the lack of an intragenic vector system [7]. We present here the development of a "foreign DNA-free" intragenic vector system, pUFCI (University of Florida Citrus Intragenic), by assembling T-DNA-like fragments with functional equivalents of T-DNA border sequences from C. clementina and recovery of positive intra-/cisgenic regenerants through PCR analysis.…”

Influence of surfactants on growth and regeneration from mature internodal stem segments of sweet orange (Citrus sinensis) cv. Hamlin

“…Despite the need for new and improved cultivars the rate of development is slow due to long juvenile period, high heterozygosity and sexual incompatibility factors (Deng and Duan 2006). Gene transfer technologies such as Agrobacterium-mediated transformation Grosser 2009, 2010;Pasquali et al 2009;Ballester et al 2010;Duan et al 2010;Dutt et al 2011), particle gun bombardment and protoplast fusion (Wang et al 2010;Grosser and Gmitter 2011) offer an alternative approach for the transfer of gene traits into important germplasms. For this to be viable, a reliable and efficient tissue culture system needs to be established for the production of regenerated shoots.…”

Recent Advances in Fruit Species Transformation

“…Apple (Malnoy et al, 2010) and pineapple sweet orange (Ballester et al, 2010) transformation using ''clean'' binary vector including only the transgene of interest were carried out to create marker-free transformants. Very recently, melon (C. melo L. cv Hetao) was transformed with a marker-free and vector-free antisense 1-aminocyclopropane-1-carboxylic acid oxidase construct via the pollen-tube pathway and transgenic lines are choosen by PCR without using any selectable marker agent (Hao et al, 2011).…”