GM Crop Database

Database Product Description

C5 (ARS-PLMC5-6)
Host Organism
Prunus domestica (Plum)
Trait
Resistance to plum pox virus (PPV)
Trait Introduction
Agrobacterium tumefaciens-mediated plant transformation.
Proposed Use

Production for human consumption.

Product Developer
United States Department of Agriculture - Agricultural Research Service

Summary of Regulatory Approvals

Country Food Feed Environment Notes
United States 2009 2009 2007

Introduction Expand

Plum pox, also known as sharka, is the most devastating viral disease worldwide of stone fruit including peaches, apricots, plums, nectarines, almonds, and sweet and tart cherries. The disease significantly limits stone fruit production in areas where it is established. More than 100 million stone fruit trees in Europe are infected. First described on plums in Bulgaria in 1915, plum pox has spread to a large part of Europe, the Mediterranean, the Middle East (Egypt and Syria), India, and Chile. In 1999, for the first time in North America, plum pox was detected in a Pennsylvania orchard.

The virus causing the disease is plum pox potyvirus (PPV). PPV infects various members of the genus Prunus, such as the stone fruit trees mentioned above. Wild and ornamental species of this genus may also become infected by some strains of the virus. Some weed hosts identified in the field and numerous hosts tested in laboratory settings have also been known to become infected with PPV.

Plum pox is economically important because it can cause fruit to be unmarketable and can decrease the yield of infected trees. The severity of the disease depends on the strain of the virus present and the susceptibility of Prunus cultivars (cultivated varieties of plants). The presence of PPV can enhance the effects of other endemic viruses infecting various species of the genus Prunus, such as prune dwarf virus, Prunus necrotic ringspot virus (causes browning), and apple chlorotic leaf spot virus (causes yellowing), resulting in still greater economic losses. In southeastern France, a newly identified strain of PPV induces severe necrosis, resulting in early leaf drop and tree decline, even in the absence of endemic Prunus viruses.

Plum pox is spread from plant to plant by several aphid vectors (insects that suck sap from plants then carry the virus to other plants) and is also transmitted in budwood used for grafting. Studies indicate that at least 14 aphid species can transmit PPV. These species include Myzus persicae, Aphis spiraecola, A. gossypii, and A. fabae. Some PPV strains have been identified that are not transmissible by aphids. Control and prevention measures for PPV include field surveys, use of certified nursery materials, use of resistant plants (when available), control of aphids, and elimination of infected trees in nurseries and orchards.

Sources of resistance exist in Prunus but are not abundant and, for this reason, a genetically engineered PPV-resistant plum has been developed using the coat protein gene from the virus itself. Multiple insertions of the gene have been identified in the plant, but no coat protein is produced by these genes. The resistance is due to RNA silencing, where the expression of a transgene induces the plant to degrade RNA of the same sequence, including any RNA from an infecting virus, thus preventing infection.

In addition to the use of the engineered variety for direct production, the resistance can by transferred through hybridization to other plum trees. This provides a unique source of germplasm for future breeding programs worldwide as there are few sources of resistance to this disease in conventional germplasm and they have proved difficult to introgress into cultivated varieties. In the United States, there are no plans at this time (2007) to release the transgenic varieties for commercial use as the disease is being controlled though quarantine practices, however discussions with breeders have initiated to develop plans for the deployment of this trait should the need arise.

Summary of Introduced Genetic Elements Expand

Code Name Type Promoter, other Terminator Copies Form
CP plum pox potyvirus (PPV) VR

CaMV 35S

A. tumefaciens nopaline synthase (nos) 3'-untranslated region

1 full length, 1 re-arranged
nptII neomycin phosphotransferase II SM nopaline synthase (nos) from A. tumefaciens A. tumefaciens nopaline synthase (nos) 3'-untranslated region 1 full length, 4 re-arranged
uidA beta-D-glucuronidase SM CaMV 35S A. tumefaciens nopaline synthase (nos) 3'-untranslated region 1 full length, 2 re-arranged

Characteristics of Prunus domestica (Plum) Expand

Center of Origin Reproduction Toxins Allergenicity

Southern Europe or Western Asia around the Caucasus Mountains and the Caspian Sea

Insect pollinated and generally self-incompatible, with both gametic and sporophytic incompatability systems.

Plum seeds contain cyanogenic glycosides, which produce cyanide when eaten.

People sensitive to birch pollen may also experience an oral allergic reaction after eating plums.

Modification Method Expand

A number of transgenic lines were produced by transformation of the plum cultivar ‘Bluebyrd’ with Agrobacterium tumefaciens strain C58/Z707 containing a binary T-DNA plasmid. Seeds of the plum cultivar were sterilised and soaked in sterile water overnight before the seed coat was removed and the cotyledons slit open. The embryonic axis was removed and the hypocotyl segment cut into slices approximately 1mm thick. These slices were immersed in a liquid culture of the the Agrobacterium strain and then cultured on medium containing various plant hormones to induce shoot formation and regeneration.

The binary plasmid was derived from pGA482GG and contains the nptII and uidA (gus) marker genes, as well as tetracycline and gentamicin antibiotic resistance genes. The nptII gene is under control of the nopaline synthase promoter (nos) and nos terminator. The uidA gene is under control of the 35S promoter and nos terminator. The tetracycline and gentamicin marker genes are under control of prokaryotic promoters and therefore are not expressed in plants. In addition to these intact genes, pGA482GG contains an interrupted ß-lactamase gene. Sequencing analysis shows that this gene is interrupted by a cos site that renders the gene non-functional. The nptII gene codes for the neomycin phosphotransferase enzyme (NPT) which provides resistance to the antibiotic kanamycin as a selectable marker. The uidA gene codes for the ß-glucuronidase enzyme (GUS) as another marker gene.

The coat protein gene of the plum pox virus containing the 35S promoter and the nos terminator, from plasmid pBIPCP was subcloned into HindIII-digested pGA482GG and the resulting plasmid was designated pGA482GG/PPV-CP-33. This plasmid was used to electrotransform A. tumefaciens strain C58/Z707 and used for transformation of plum tissue.

Characteristics of the Modification Expand

The Introduced DNA

Information from Southern blots indicated that there are multiple copies of the T-DNA present in C5. Cloning and sequencing of genomic clones, together with PCR analysis, has revealed five inserted fragments all in close proximity and which behave as a single genetic locus. One of the fragments contains a complete insert, consisting of the PPV coat protein gene, the nptII gene and the uidA gene. The other four fragments contain only partial insertions with deletions, duplications and rearrangements observed. One fragment is comprised of an inverted repeat of the PPV coat protein gene, which is thought to be critical in generating the resistance trait.

Genetic Stability of the Introduced Trait

The structure of the inserted DNA has been evaluated in both vegetatively propagated plants and progeny of C5 with no changes noted, indicating that the locus is stable. In addition, expression of the resistance trait has been maintained during seven years of field trials with C5 clones grafted onto rootstocks, as well as in vitro and pot-grown seedlings of C5 crossed with cultivated varieties of plums. The plants remained free of virus despite very high inoculation pressure obtained by grafting PPV infected material onto the transgenic shoots and grafting transgenic shoots onto infected rootstocks.

Using assays for the uidA gene product, together with PCR assays, the progeny of crosses made with C5 showed close to a 1:1 segregation pattern, indicating that the integrated DNA behaved as a single locus and was inherited in a Mendelian fashion.

Expressed Material

The nptII and uidA genes produce the NPT and GUS enzymes, respectively, in C5. Expression of these proteins was demonstrated not only by resistance to Kanamycin, provided by the NPT protein, but also by assays for the GUS protein using the colorimetric substrate, X-Gluc which is converted into a blue precipitate by the enzyme.

There is no detectable protein produced by the PPV coat protein gene and assays for the messenger RNA from this gene were also negative, prompting the deduction that the resistance mechanism is through post-transcriptional gene silencing (PTGS). This was confirmed by nuclear run-on analysis which confirmed that mRNA from the PPV coat protein was being produced, but was not accumulating in the plant cells. Another characteristic of PTGS is the production of short interfering RNA molecules (siRNA) that are derived from double-stranded RNA degraded by a specific RNAseIII-like enzyme referred to as DICER. It is these siRNA molecules which are thought to provide the specificity of PTGS in silencing certain genes. Detection of siRNA specific to the PPV coat protein genes confirmed this mechanism of transgenic virus resistance in C5.

Environmental Safety Considerations Expand

Field Testing:

Field trials were established in 1996-1997 in Poland, Spain and Romania with C5 shoots grafted onto non-transgenic rootstocks. These trials were maintained for efficacy testing of the transgenic lines and deliberately inoculated with PPV using infected grafts or interspersed with infected trees to test for resistance to aphid transmitted virus. Further field trials in the United States were approved in 1995 and included trees which were rooted from C5 clones as well as grafted material. Pollen movement from this trial has been monitored between blocks of transgenic and non-transgenic trees.

Outcrossing:

Outcrossing is expected in P. domestica due to the high levels of self incompatibility shown in this species. Seeds collected from open pollinated field trials have all shown a 1:1 segregation pattern of transgenic to non-transgenic, indicating that C5 is not self-pollinating nor being pollinated by other transgenic lines. Non-transgenic control trees of the same parental material in the experimental blocks have not produced any transgenic progeny, confirming that this line is self-incompatible and the transformation process has not altered the compatibility status of the material.
Seeds collected from non-transgenic trees planted approximated 500 and 1000 meters from the transgenic plot were sampled over six years. Of 2,950 seeds tested, only two transgenic seeds were identified, from the 500 meter plot (outcrossing frequency was 2 in 850 seeds – 0.23%). It was considered that bees were the most likely vehicle of pollen movement. Further experiments with nine ‘sentinel’ sites are being continuously monitored.

Gene flow between P. domestica and other Prunus species is unlikely as these species are nearly all diploid while P. domestica is hexaploid. Successful crosses have been made between P. domestica and other species, but most hybrids were sterile and the literature contains many references to the difficulty of interspecific hybridisation within Prunus.
Introgression between wild and cultivated species is not documented, but is physically possible as escapes of cultivated varieties are frequently found in woods, pastures and abandoned orchards. However, intercrosses with truly wild species is unlikely as these species are extremely different in morphology as well as in adaptation.

Weediness potential:

P. domestica is not described as a weedy species and none of the Prunus species that may be sexually compatible with P. domestica are described as weedy species. In addition, plum is not listed as a noxious weed by the United States Federal authorities or on other weed lists.

Secondary and Non-Target Effects:

The biology of the C5 plum trees with respect to their potential to affect non-target organisms such as beneficial insects (including pollinators such as bees), and biocontrol organisms was evaluated. The C5 plum does not express detectable coat protein from PPV, which eliminates concern of protein exposure to non-target organisms. Furthermore, even if C5 did express viral coat protein this would not increase the issue of potential impacts to non-target organisms as the PPV coat protein is not known to have any toxic properties. Plant viruses are ubiquitous in the environment and cause damage to fruits, leaves, seeds, flowers, stems, and roots of many important crop species. These viruses infect virtually every plant species and, under natural conditions, certain plant viruses are nearly always present on particular crop or weed hosts. Viral coat proteins are therefore routinely ingested by virtually all mammals when virus-infected fruits and vegetables are consumed. The small-interfering RNAs (siRNA) responsible for the PTGS resistance mechanism in C5 plum are also not of concern as nucleic acids are a normal part of every living organism and do not have toxic or allergenic properties.

The nptII and ß-glucuronidase genes are commonly used marker genes found in soil inhabiting E. coli bacteria. These bacteria are not plant or human pathogens, and do not cause disease symptoms or the production of infectious agents in plants. In addition, these marker genes are not known to cause adverse effects to non-target organisms and both have been granted exemption from the requirement of a tolerance by EPA for use in or on all raw agricultural commodities

Impact on Biodiversity:

Analysis of available information indicates that C5 plum exhibits no traits that would cause increased weediness, that its unconfined cultivation should not lead to increased weediness of other cultivated plum or other sexually compatible relatives, and that it is unlikely to harm non-target organisms common to the agricultural ecosystem or threatened or endangered species. Based on this analysis, there is no apparent potential for significant impact to biodiversity.

Analysis of data on agronomic performance, disease and insect susceptibility, and compositional profiles of the plums indicate no significant differences between C5 plum and non-transgenic counterparts that would be expected to cause either a direct or indirect plant pest effect on any raw or processed plant commodity. Data supplied by the applicant, including results of 10 years of field tests in various environments, suggest that the C5 plum trees have not had observable or measurable impacts on the ecosystems in which they have been allowed to grow. Based upon available information, it was determined that there are no past, present, or reasonably foreseeable actions that would aggregate with effects of the proposed action to create significant cumulative impacts or significantly reduce the long-term productivity or sustainability of any of the resources (soil, water, ecosystem quality, biodiversity, etc.) associated with the ecosystem in which C5 plum is planted.

Abstract Collapse

The genus Prunus comprises a diverse group of plants with many botanical species that have been cultivated for the last 3000 years and contains approximately 400 species of trees and shrubs. Many species and cultivars are grown for their edible fruits including almonds, apricots, cherries and peaches, in addition to plums and prunes. Other species are planted for their ornamental value and a few are commercially important for hardwood timber production.

Plum species are found native throughout the Northern hemisphere and the most important commercial species of plums are generally classified in two groups, the European plums (Prunus domestica L.) and related forms with a hexaploid chromosome number (2n=6x=48) and the Japanese plums (Prunus salicina) and their hybrids with diploid chromosome numbers (2n=2x=16). P. domestica is believed to have originated as a natural alloploid between P. cerasifera and P. spinosa and seems to have originated in Southern Europe or Western Asia around the Caucasus Mountains and the Caspian Sea.

World production of all plums was over nine million tons in 2005, with China accounting for nearly half of this total. Of the other countries in the top 10 producers, only the United States and Chile are from outside Europe. Within the United States, California is the leading producer of dried plums, accounting for 70% of the world exports for this commodity.

Plum pox virus (PPV) is the causal agent of Sharka disease, the most serious virus disease of stone fruits. PPV is spread from tree to tree by aphids and through infected budwood used for grafting. Symptoms of PPV infection include leaf and fruit yellowing, fruit deformation, premature fruit drop and tree decline. PPV has spread throughout Europe where it has destroyed over 100 million stone fruit trees and has also spread to India, Egypt, Lebanon, the Azores, Chile and has also been found in the US, where there is a strict quarantine and eradication program in Pennsylvania. Few highly resistant Prunus varieties have been developed through conventional breeding and thus genetically engineered resistance is considered to be an important option.

C5 is a genetically engineered plum clone containing the PPV coat protein gene that has been found to be highly resistant to PPV. The mechanism of resistance has been determined to be through post-transcriptional gene silencing as there is no protein expressed from the inserted coat protein gene. The resistance has proven to be highly stable, with field tests carried out over eight years in three European countries where the virus is present. No C5 trees in any of these field tests have become infected through natural aphid inoculation and graft inoculated trees show few and mild symptoms of the disease.

In addition to the PVP coat protein gene, C5 contains the nptII gene for resistance to the antibiotic Kanamycin as a selectable marker and the gene coding for the GUS enzyme, which was used as an assayable marker for the presence of the transgene. C5 was produced by Agrobacterium-mediated transformation of hypocotyl slices. Molecular analysis of C5 identified five separate transgene insertions, of which one is a complete insert and the others are either partial or re-arranged fragments of the T-DNA.

C5 has been developed as a pre-emptive approach to the control of PPV should the disease become established in the United States. The are currently (2007) no plans to release this event commercially, but the event is available should the need arise and the resistance trait expressed in the event is available to breeders to improve other varieties of plum.

Links to Further Information Expand


This record was last modified on Tuesday, September 15, 2015