GM Crop Database

Database Product Description

Vector 21-41
Host Organism
Nicotiana tabacum (Tobacco)
Trait
Nicotine reduced.
Trait Introduction
Agrobacterium tumefaciens-mediated plant transformation.
Proposed Use

Production for processing into cigarettes.

Product Developer
Vector Tobacco Inc.

Summary of Regulatory Approvals

Country Food Feed Environment Notes
United States 2002

Introduction Expand

Tobacco line Vector 21-41 was developed to reduce the presence of nicotine in the leaves of commercial tobacco (Nicotiania tabacum). Vector 21-41 delivers nicotine levels around 20 times less than conventional tobacco. Nicotine is commonly regarded as the addictive substance in tobacco and is a precursor of Tobacco Specific Nitrosamines which are mutagenic and carcinogenic substances normally found in tobacco. It is thought that delivering nicotine at levels well below those determined to be addictive can reduce dependency on tobacco products.

The synthesis of nicotine is isolated in the roots of tobacco and subsequently transported via the phloem to the leaves. Nicotine is the product of two enzymatic pathways: the methylpyrroline pathway, which produces the nicotine precursor N-methylpyrrolinium cation; and the pyridine nucleotide cycle, which produces the precursor nicotine acid.

In the methylpyrroline pathway, L-ornithine is converted via ornithine decarboxylase to putrescine which in turn is acted on by putrescine N-methyltransferase (PMTase) to produce 4-methyle putrescine and subsequently N-methylpyrrolinium cation. PMTase is the limiting enzyme in this pathway.

The pyridine nucleotide cycle begins with 3-phosphoglyceraldehyde which condenses with aspartic acid, to form quinolinic acid. Quinolinic acid is subject to the action of quinolinic acid phosphoribosyltransferase (QPTase) and through a series of steps becomes nicotinic acid. In this pathway, QPTase is the limiting enzyme.

In Vector 21-41 the pyridine nucleotide cycle was down-regulated through the insertion of the antisense configuration of the gene coding for QPTase (NtQTP1). The reduction in expression of QPTase resulted in a dramatic decrease in the production of nicotinic acid and thus nicotine.

Summary of Introduced Genetic Elements Expand

Code Name Type Promoter, other Terminator Copies Form
EC2.4.2.19 nicotinate-nucleotide pyrophosphorylase (carboxylating) NR NtQPT1 promoter A. tumefaciens nopaline synthase (nos) 3'-untranslated region
nptII neomycin phosphotransferase II SM

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

Characteristics of Nicotiana tabacum (Tobacco) Expand

Center of Origin Reproduction Toxins Allergenicity

The species is indigenous to the Americas.

Flowers are generally self-pollinated, but insects can facilitate cross-pollilnation.

Nicotine, a highly toxic substance, is synthesized in the roots of the tobacco and transported to the leaves.

Exposure to tobacco plants has been observed to cause allergic symptoms in people with other plant allergies.

Modification Method Expand

Tobacco line Vector 21-41 was produced by Agrobacterium-mediated transformation of leaf discs from Burley 21 LA using the binary vector pYTY32. Transformed plantlets were regenerated on MS medium containing kanamycin as a positive selection agent.

The plasmid vector pYTY32 was transformed into disarmed Agrobacterium tumefaciens strain LBA 4404 via electroporation. The plasmid pYTY32 was a modification of the pBin19 plasmid which and carried the NtQTP1 promoter and the antisense configuration of NtQTP1 cDNA As well, the plasmid T-DNA region contained the nptII gene coding for kanamycin resistance as a marker. Transcription of the nptII gene was regulated using the promoter and termination sequences from the A. tumefaciens nopaline synthase encoding gene (nos).

Characteristics of the Modification Expand

The Introduced DNA

The inserted DNA was characterized using a combination of restriction enzyme digestion and Southern blot labelling to confirm the number of sites of insertion of the pYTY32 T-DNA, and polymerase chain reaction (PCR) amplification using specific primer pairs to confirm gene organization and integrity. PCR amplification was also used to test for the incorporation of plasmid-derived sequences outside of the T-DNA region.

The presence of an endogenous NtQPT1 gene in tobacco necessitated the use of a probe for the marker gene nptII as opposed to a probe for the NtQPTII antisense transgene. Southern Blot analysis confirmed the presence of two T-DNA insertions segregating as a single locus. The transgene conferring kanamycin resistance was found to segregate as a single locus and be co-segregated with the low nicotine phenotype. DNA blot analysis data demonstrated that Vector 21-41 T1 progeny carried 2 copies of the transgene.

Between 2.1 kbp and 2.4 kbp of backbone pYTY32 sequences that flanked the T-DNA left border were also transferred to Vector 21-41, however these did not include any complete coding regions for any genes.

Genetic Stability of the Introduced Trait

Segregation and stability data indicated two copies of the T-DNA region of pYTY32 at a single locus. The stability of the insertion was demonstrated through multiple generations derived through self-pollination. Vector 21-41 was derived from the T1 generation and stable insertion of the insert was demonstrated through Southern blot analysis of T1 progeny as well as low nicotine expression in subsequent generations.

Expressed material

The production of nicotine in Vector 21-41 was down-regulated in the roots of the plant by an antisense strategy. In this case, no new polypeptide was expressed, aside from the NPTII enzyme. QPTase activities are thus decreased in Vector 21-41 as compared to Burley 21LA.

Environmental Safety Considerations Expand

Outcrossing

Tobacco is not considered a likely candidate for any significant level of outcrossing. The plant is generally self-pollinated being possessed of tubular flowers and possible insect vectors travel only short distances. In the commercial production of tobacco, flowers are generally removed prior to anthesis and are thus not allowed to shed pollen further decreasing the risk of outcrossing. Tobacco producers do not save seed and certified seed production of Vector 21-41 will occur only in isolation.

N. trigonophylla, N. repanda and N. bigelovii are indigenous Nicotiania species that are found in the U.S. and are cross-compatible with N. tabacum. None of these species grows in areas in which commercial tobacco production take place.

Weediness potential

No competitive advantage was conferred to Vector 21-41 by the transformation. The plants were not more or less resistant to insect predation than standard tobacco. Tobacco has not shown any potential to become a noxious weed.

Secondary and Non-Target Adverse Effects

The presence of nicotine in tobacco is thought to have evolved as a defense against insect predation. It is known that insects have developed resistance to nicotine, resulting in the need for multiple applications of pesticides on conventional tobacco crops. Vector 21-41 was found to have similar yields to other low alkaloid tobacco cultivars when a standard package of pesticides was applied. With no insect management both Vector 21-41 line and conventional tobacco had no commercial yield when faced with heavy insect pressure. Vector 21-41 did not result in increased pesticide application.

Impact on Biodiversity

There is no expected impact on biodiversity from Vector 21-41 tobacco. The line would not be grown outside the areas currently host to tobacco production and the possibility of outcrossing is extremely remote.

Other considerations

The parent plant Burley 21 LA (low alkaloid) has not been grown for commercial production for some time. The cultivar has been found to have a high susceptibility to blue mold, as does Vector 21-41. As well, the parent line is susceptible to insects and this trait is also noted in Vector 21-41, however, neither line requires more stringent pest control than is normally used in tobacco cultivation.

Abstract Collapse

Tobacco (Nicotina tabacum) was grown commercially in 127 countries in 2004, with a combined production of 6.5 million metric tonnes. The major producers of tobacco were China, Brazil, India, the United States, Zimbabwe, Turkey and Indonesia. This cultivated plant is thought to be a hybrid of N. sylvestris and N. tomentosiformis. All Nicotiana species are characterized by the presence of nicotine and other alkaloids.

Tobacco is processed into cigarettes, cigars, snuff, and chewing tobacco. Tobacco is widely regarded as being highly addictive due to the presence of high (20000 ppm to 30000 ppm) levels of nicotine in conventional varieties. It is generally agreed that reducing the level of nicotine delivered by tobacco products will reduce dependence on tobacco, which is commonly regarded as large contributor to cancerous diseases and premature mortality worldwide.

The transgenic tobacco line Vector 21-41 was developed to reduce nicotine levels below those thought to be addictive. Vector 21-41 routinely contains 20 fold less nicotine than conventional varieties and also produces 15 fold less mutagenic and carcinogenic Tobacco Specific Nitrosamines. An antisense strategy was used to downregulate expression of the enzyme responsible for the production of nicotinic acid, a nicotine precursor. Specifically the antisense configuration of NtQPT1 a gene coding for quinolinic acid phosphoribosyltransferase (QPTase) was inserted into Burley 21 LA (a low-alkaloid variety) via Agrobacterium-mediated transformation.

Vector 21-41 was field tested in the U.S. and Argentina. Vector 21-41 did not prove to be substantially different than the parental line: Burley 21 LA or Burley 21, in terms of agronomic performance. Vector 21-41 did exhibit susceptibility to fungal diseases such as blue mold and insects, but this was not substantially different from the parental line and could be controlled by conventional practices.

In field trials, Vector 21-41 leaves had 700 to 1500 ppm nicotine, while conventional varieties grown in the same conditions produced 10000 to 30000 ppm nicotine. The percentage of reducing sugars was increased in Vector 21-41 compared to conventional varieties almost two-fold.

Due to the nature of the tobacco plant and cultivation methods currently used, it was not thought that Vector 21-41 posed an undue environmental risk. Tobacco is usually a self-pollinating plant, being possessed of tubular flowers and not prone to wind pollination. As well, out-crossing with related species is rare and these species are not generally found in areas where tobacco is grown.

Cultivation of tobacco is not conducive to outcrossing. Most growers do not save tobacco seed, preferring to rely on certified seed for planting. As well, in commercial production, the flowers are removed prior to anthesis, pollen shed or seed development to encourage leaf growth. Vegetative reproduction in tobacco is rare in field conditions.

An antibiotic resistance marker gene (nptII) encoding the enzyme neomycin phosphotransferase II (NPTII), which inactivates aminoglycoside antibiotics such as kanamycin and neomycin, was also introduced into the genome of these plants. This gene was derived from a bacterial transposon (Tn5 transposable element from Escherichia coli) and was included as a selectable marker to identify transformed plants during tissue culture regeneration and multiplication. The expression of the nptII gene in these plants has no agronomic significance and the safety of the NPTII enzyme as a food additive was evaluated by the United States Food and Drug Administration in 1994 (US FDA, 1994).

Vector 21-41 is not expected to impact biodiversity or to become a weed nuisance. The transformation does not convey any competitive advantage to the plant nor does it allow the production of tobacco outside areas currently under tobacco cultivation. It has no known adverse effects on animals or insects feeding on the plant.

Links to Further Information Expand

U.S. Department of Agriculture, Animal and Plant Health Inspection Service U.S.Department of Agriculture, Animal and Plant Health Inspection Service

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