ER-AF-N04-2: APPLICATION FOR APPROVAL TO FIELD TEST IN CONTAINMENT A GENETICALLY MODIFIED ORGANISM
TITLE: Field testing of potatoes genetically modified for disease resistance Applicant details
1. Dr Tony Conner
New Zealand Institute for Crop & Food Research Ltd Private Bag 4704
Christchurch
Telephone: +64 3 3256400
2. Street address (for courier delivery): Gerald St, Lincoln, Canterbury 3. Contact person:
Tony Conner, Scientist Facsimile: +64 3 3252074 Telephone: +64 3 3256400 e-mail: connert@crop.cri.nz 4. Executive summary
Over the next 5 years the New Zealand Institute for Crop & Food Research Ltd is intending to conduct a series of small-scale contained field trials on potato plants. These plants have been subjected to a genetic engineering process in order to make them resistant to diseases such as bacteria inciting soft rot diseases. This target character is a major problem for the New Zealand potato industry. Breeding for resistance is limited by a lack of appropriate germplasm, and current control measures often heavily rely on pesticide use. The transfer of genes from unrelated organisms offers a novel, broad spectrum approach to improving disease resistance in crop plants. The main aim of this trial is the selection of genetically modified potato lines with genes anticpated to confer increased disease resistance which will offer benefits such as environmental enhancement and sustainability by reducing pesticide residues in soils and ground water, as well as enhancing genetic diversity within potato germplasm.
The introduced DNA sequences are combinations of DNA from viruses, bacteria, animals and other plants that have been artificially modified to maximise their activity in plants.Three genetic engineering approaches are being used to target improved resistance to soft rot bacteria. These involves the development and transfer of genes encoding three peptides: cecropin (artificial DNA sequence based on peptides from silkworms), magainin (artificial DNA sequence based on peptides from toads), and lysozyme (ex T4 bacteriophage); all of which are highly active against Erwinia isolates inciting disease on potatoes. The disease resistance genes are attached to a marker gene conferring resistance to the antibiotic kanamycin which is used to select engineered plant cells from non-engineered cells during the genetic engineering process. DNA sequences involved in directing the transfer of the novel genes into potato were derived from the soil bacterium, Agrobacterium tumefaciens. Other DNA sequences controlling the activity of the novel genes/peptides in potatoes were derived from cauliflower mosaic virus, alfalfa mosaic virus, Agrobacterium tumefaciens, tobacco, and barley.
Over the 5 year period, the proposed field trials will consist of single plots of ten plants for up to 150 genetically engineered lines each year, plus appropriate control plots of the non engineered original potato cultivar. The lines for field testing will be selected on the basis of the presence of specific new genes and normal plant appearance as determined from laboratory studies and evaluation in containment greenhouses at Lincoln. Each year a detailed field plan and list of lines will be sent to the Authority prior to planting.
Well characterised containment systems, based on those successfully used in similar trials on genetically modified potatoes at Crop & Food Research over the past decade, will minimise the main risk, that of dispersal of the transgenic material. The transgenic plants will be surrounded by a buffer zone of 3 rows of non-transgenic potato plants and an isolation distance of at least 50 metres from other non-modified potato crops will virtually eliminate any opportunity of gene escape through pollen dispersal. Subsequent monitoring of the trial site for 1-2 years after harvest, coupled with the immediate removal of any potato plants, will ensure the complete elimination of genetically engineered potato plants from the trial site. There is no confidential information included as part of this application.
Organism details
5. The identification of the organism
Taxonomic name: The parent organism is potato (Solanum tuberosum L.), a member of the family Solanaceae. The potato originated in the Andes in Peru and Bolivia from where it was introduced into Europe and later into New Zealand. The potato is distributed throughout the world and as a cultivated crop ranks fourth behind wheat, maize and rice in area planted worldwide. In New Zealand potatoes are grown throughout the country, both as an agricultural crop on approximately 10,000 ha (Dunbier and Bezar 1995) and in domestic gardens.
Characteristics: Naturalised populations of potato are known in New Zealand, and commonly occur on waste land, rubbish dumps, around old dwellings, camp sites, and coastal beaches frequented by people (Webb et al. 1988). Although such escapes from cultivation are frequently observed, they are only considered as causal or transient escapes and the species is not considered as being fully naturalised (Webb et al. 1988).
The potato survives primarily through tubers and rarely by seed under cultivated conditions.
The plant does not tolerate frost or high temperatures and the tubers will rot under anaerobic conditions. It is not an aggressive competitor outside the usual cultivated situation. Gene transfer between plants may occur through pollen, however the intended field trial will be isolated to eliminate the possibility of pollen transfer to other potato crops (see 15).
Potato seedlings rarely establish the following year because of weed competition and the ease of removal during cultivation. We intend to collect all berries that develop on the plants in the proposed field trial (see 15). Potatoes have not been reported to naturally cross with any other Solanaceous species naturalised in New Zealand (see 14).
This field trial will involve transgenic lines of the Iwa cultivar. The use of Iwa by the New Zealand potato industry is declining, but it is highly amenable to Agrobacterium-mediated transformation (Conner et al. 1991b). Iwa was released in 1976. It was bred by the former Crop Research Division of DSIR in New Zealand from a cross between 119-224 and a Sebago x Harford hybrid. Iwa is a late main crop potato with smooth yellow skin and white flesh. It has good boiling quality and good flavour.
Name of the organism for the Authorities public registrar:
potato (Solanum tuberosum L.)
6. Details of the genetic modifications
Vectors with genes targeting soft rot bacteria
The DNA inserted was a modified T-DNA of Agrobacterium tumefaciens, carried on the binary vectors pSHIVA, pBINPMgA-D, pBINPLys. These binary vectors are illustrated in Figure 1.
The pSHIVA vector was constructed by Dr Andrew Gleave (HortResearch, Mt Albert) and is based on the binary cloning vector pART27 (Gleave 1992). The remaining vectors were constructed by Ms Philippa Barrell (Crop & Food Research, Lincoln) and are based on another binary cloning vector, pBINPLUS (Engelen et al 1995). All vectors contained the left and right borders of the Ti plasmid from Agrobacterium tumefaciens, between which are two genes that can be expressed in plants:
(i) A chimeric gene conferring kanamycin resistance to plant cells consisting of the coding region of neomycin phosphotransferase II from the bacterial transposon Tn 5 inserted between the promoter and polyadenylation signals from the nopaline synthase (nos) gene of Agrobacterium tumefaciens.
(ii) one of the following antibacterial genes:
• a lysozyme gene (ex bacteriophage T4) (pBINPLys);
• a synthetic potato gene that encodes an analogue of cecropin B (ex giant silk moth), designed and artificially constructed using potato codon usage (pSHIVA); and
• a synthetic potato gene that encodes magainin II (ex African clawed frog), also designed and artificially constructed using potato codon usage. Four magainin II genes have been developed -
A: encodes the native magainin II peptide (pBINPMgA);
B: has a point mutation that converts the 7th amino acid residue from histidine to arginine (to increase peptide stability in plants) (pBINPMgB);
C: has three point mutations that convert the 8th, 13th and 18th amino acid residues to alanine (to increase antibacterial activity) (pBINPMgC);
D: combines both sets of point mutations from B and C above (pBINPMgD).
The chimeric lysozyme gene consists of a 35S promoter from CaMV, a barley α-amylase signal peptide for targeting to the intercellular space, the coding region of the bacteriophage T4 lysozyme gene, and a 35S polyadenylation signal from CaMV. The cecropin B analogue (Shiva) and magainin II coding regions are cloned downstream of the 35S promoter from cauliflower mosaic virus (CaMV), a synthetic sequence encoding an untranslated leader sequence from alfalfa mosaic virus, and a N-terminal translational fusion to a synthetic sequence encoding a signal peptide from the tobacco extracellular PR-S protein (Sijmons et al. 1990). This signal sequence targets these antibacterial peptides to the intercellular space. These two chimeric genes have a polyadenylation signal from the nopaline synthase (nos) gene of Agrobacterium tumefaciens.
Binary vector construction
The pSHIVA vector was constructed by isolating the chimeric cecropin B analogue gene as a 1.2 kb NotI fragment and ligating it into the unique NotI site within the lacZ' region of the binary vector pART27 (Gleave 1992). Similarly, the binary vectors pBINPMgA, pBINPMgB, pBINPMgC, and pBINPMgD were constructed by isolating the chimeric magainin II gene as a 1.2 kb EcoRI-HindIII fragment and ligating it into the unique EcoRI and HindIII sites of the lacZ' region of the binary vector pBINPLUS, an improved derivative of pBIN19 (Engelen et al. 1995).
For the pBINPLys vector the chimeric lysozyme gene was isolated as a 1.3 kb HindIII fragment from pSR8-1, a precursor to pSR2-5 (Düring et al. 1993) and cloned into the unique HindIII site of binary vector pBINPLUS (Engelen et al. 1995). A map of the vectors is presented in Figure 1.
Transfer of binary vectors to Agrobacterium
All binary vectors were introduced into the disarmed Agrobacterium tumefaciens strains by the freeze-thaw method (Hofgen and Willmitzer 1988). Either strain LBA4404, strain AGL1 or strain EHA105 were used. LBA4404 has the chromosomal background of strain ACH5 and harbours the plasmid pAL4404 which is a non-oncogenic derivative of pTiAch5 from which the T-DNA has been deleted (Hoekema et al. 1983). AGL1 has the chromosomal background of strain C58 with a recA mutation and harbours the plasmid pTiBo542-T which is a non-oncogenic derivative of pTiBo542 from which the T-DNA has been deleted (Lazo et al.
1991). EHA105 also has the chromosomal background of strain C58 and harbours the plasmid pEHA105 which is a non-oncogenic derivative of pTiBo542 from which the T-DNA has been deleted (Hood et al. 1993). All three of these Agrobacterium strains therefore carry an intact vir region and can mediate the introduction of any T-DNA present in the bacteria into plant cells (in this case the modified T-DNA of the binary vectors).
Gene transfer to potato
All the transgenic potato lines were produced essentially following our standard potato transformation protocols (Conner et al. 1991b). Leaf segments of in vitro grown potato plants were dipped briefly in a suspension culture of Agrobacterium harbouring the binary vector. After blotting dry on filter paper they were cultured on potato callusing medium (MS medium [Murashige and Skoog, 1962] plus 500 mg/l casein hydrolysate, 40 mg/l ascorbic acid, 0.2 mg/l NAA and 2 mg/l BAP). Two days later the leaf segments were transferred to the same medium supplemented with 200 mg/l Timentin (to inhibit overgrowth by the Agrobacterium cells). After five further days, the leaf segments were transferred to the callusing medium plus 200 mg/l Timentin (to inhibit overgrowth by the Agrobacterium cells) plus 50 mg/l of kanamycin (to select for transformed cell colonies). After 3-6 weeks of growth, kanamycin-resistant callus colonies were subcultured onto regeneration medium (MS medium plus 500 mg/l casein hydrolysate, 40 mg/l ascorbic acid, 5 mg/l GA3, 1 mg/l zeatin, with sucrose reduced to 0.5%) supplemented with 100 mg/l Timentin and 50 mg/l kanamycin for further growth and selection. A single regenerated shoot from each cell colony was transferred to MS medium plus 500 mg/l casein hydrolysate, 40 mg/l ascorbic acid and 100 mg/l Timentin to micropropagate plants.
Stability and integrity of the transferred genes
The transgenic potato lines have been selected for their resistance to kanamycin which is a consequence of the expression of the neomycin phosphotransferase II gene. Following plant regeneration and 5-6 cycles of in vitro cloning, all independently selected transgenic lines to be field tested will have exhibited resistance to kanamycin as defined by the ability to initiate roots on micropropagation medium supplemented with 50 mg/l kanamycin.
Only the modified T-DNA of the binary vectors is transferred to plant cells. Molecular analysis using the polymerase chain reaction (PCR) will have confirmed that all lines to be field tested contain the expected fragment of transferred genes. It is not possible to determine the nature of inheritance and genetic stability of transgenes in potato since plants do not usually flower when grown in our containment greenhouse.
Desired characteristics
The antibacterial genes being used are specifically designed to target Erwinia carotovora isolates known to induce bacterial soft rot. However, due to the broad spectrum activity of the peptides against a wide range of bacteria and fungi (Jaynes et al. 1987, Zasloff et al. 1988; Wilson and Conner 1995), these transgenic lines can be expected to have improved resistance to a range of pests and diseases.
7. The reason why the application is necessary
Potato is not a prohibited organism under the second schedule of the HSNO act. An application to ERMA for the proposed field trials is necessary since the potato plants have been genetically modified by the insertion of genetic material developed by in vitro techniques.
8. The nature and method of the field trials and the experimental procedures to be used Location and duration of the trials
The proposed field trials will be conducted in isolation from other potato crops at the New Zealand Institute for Crop & Food Research, Lincoln. As shown in Figure 2, the trial site has been chosen to be both easily and closely supervised, as well as inconspicuous and relatively inaccessible to the general public. The surrounding environment will consist of trial areas of experimental genotypes from a wide range of agricultural and horticultural crops such as cereals, peas, and brassicas. Annual field trials will be planted from the 1998/99 summer for five years until the 2002/03 summer and will be rotated around the site shown in Figure 3.
Associated facilities
Full research facilities for conducting the proposed field trial will be available, including cultivation, spraying and irrigation equipment associated with normal maintenance of field trials on an Agricultural Experimental Station. The field trial will also be supported by well equipped genetics, tissue culture, and molecular biology laboratories (Figure 3).
Details of the target ecosystem
The site for the proposed field trial has been maintained under rotation of a range of arable, vegetable and pasture crops since at least 1926 when it was first occupied by the former DSIR.
It is not considered to be vulnerable to any disturbances. The area where the trial will be planted is not subject to extreme conditions such as flooding or fires. Contingency plans to cope with such extreme conditions are unnecessary, since if they occurred they would effectively render the transgenic potato plants inviable.
Full trial design and experimental plan
For this proposed field trial, establishment of plots involving single rows of ten plants for up to 150 lines, is required to produce tubers for disease resistance screening. Rows of the potato plants will be spaced 75 cm apart, with 30 cm between plants within rows and a 1 m gap between plots. A proposed layout of the field trial is illustrated in Figure 4. Over the 5 year period, new lines generated by transformation with the same vectors (Figure 1) will be tested and based on previous trials, some lines may be retested and, on some occasions, the number of lines tested maybe below the maximum 150 stated. In addition, the exact shape and size of the trial site may vary from year to year to accommodate any difficulties experienced with soil conditions during previous trials. So, prior to planting the field trial each year, a list of all lines to be field tested and the final field plan will be forwarded to the Authority.
Several plots of one-two transgenic lines previously field tested and non transgenic control lines will be dispersed over the trial area to allow cross referencing both within the trial and between the different years. In the future, an additional application may be submitted to ERMA should further scale up trials be required for promising lines identified in the course of this proposed field trial.
The experimental area will be completely surrounded by 3 buffer rows of non-transgenic potato to prevent "edge effects" during the trial. These buffer rows will be planted with a potato genotype having purple tubers, to allow them to be readily distinguished from the white or red tubers of the transgenic potato lines. The field trial will be run alongside another trial testing genetically modified potato with increased pest resistance to potato tuber moth (Figure 4; see separate application). The buffer zone between the two trials will be 3 rows of non-transgenic potato plants. It should be noted, that although the exact shape and size of the trial may change over the 5 year period, the isolation distances and containment features will not. The following data will be collected from the trial:
(i) observations on the general phenotype of the transgenic lines;
(ii) the number of tubers and weight of tubers per plant;
Other matters
The capability of the transgenic potatoes to disperse and survive is fully discussed in sections 11-15. The specific potential effects being tested are well outlined under aims of the field trials in section 9. Most tubers will all be transported to the containment greenhouse facility in secure sacks for counting and weighing of tubers along with bioassay tests for tuber moth resistance.
Potato tubers used in the bioassays will be disposed of by autoclaving. Excess tubers from the transgenic plants will be disposed of by complete submersion in water for 3 to 4 days followed by burial alongside the trial site. Our experience in previous field trials indicates that this is the
most efficient way to kill and then dispose of potato tubers. There is a large concrete trough available on the Crop & Food Research experimental farm for submerging the potatoes. As outlined in section 15, both the trial and burial sites will be monitored for any survivors.
9. Purpose of the field testing for which approval is sought
Approval is sought under section 39 1b of the HSNO Act to field test potato lines genetically modified with genes anticipated to confer disease resistance.
Importance of field trials on transgenic potatoes
Field trials approved by the GMO Interim Assessment Group (IAG) on transgenic potatoes at Lincoln since 1988 have established that a significant proportion (40%) of transgenic potato lines showed unusual shoot morphologies and/or had very poor yields (Conner et al. 1991a; Conner et al. 1994). Often only about one third of transgenic lines have tuber yield approaching that of the parent cultivar. Sometimes these phenotypic changes only became apparent during the field trial and were not evident under contained greenhouse conditions or in tissue culture. Such changes have been attributed to somaclonal variation - genetic and/or physiological changes associated with the cell culture and regeneration phase of plant transformation (Conner et al.
1994). These results establish the importance of field testing many independently derived transgenic lines to identify those with agronomic performance approaching that of the parental cultivar.
Importance of soft rot resistance in potato
Soft rot diseases, incited by Erwinia bacteria, are a major problem for the New Zealand potato industry, since there are no chemical control methods available (Wright et al. 1991). Breeding for resistance is limited by the lack of appropriate germplasm. The transfer of genes encoding antimicrobial peptides offers a novel, broad spectrum approach to improving disease resistance in crop plants.
At Crop & Food Research we have genetically modified the New Zealand potato cultivar Iwa with three antibacterial genes. This had involved the development and transfer of chimeric genes encoding three peptides: cecropin B, magainin II, and T4 lysozyme; all of which are highly active against Erwinia isolates inciting disease on potatoes (Destefano-Beltran et al. 1990; Wilson and Conner 1995). The cultivar Iwa is being primarily used in these studies because it is very amenable to transformation (Conner et al. 1991b) and is highly susceptible to Erwinia soft rots (Wright et al. 1991). Some of these lines have been grown in the containment greenhouse and the resulting tubers produced have been tested for soft rot resistance. Several of the lines exhibited significantly reduced tuber rot, however the results from such bioassays are not always reliable when using tubers from container grown plants. The uniformity in the physiology and development of tubers grown in Planter Bags is poor, and such factors can markedly affect the outcome of pathogen bioassays. More meaningful resistance tests require the production of field grown tubers.
The aims associated with the field testing of the potato lines targeting soft rot resistance are:
(i) to produce field grown tubers from the transgenic lines for bioassays against bacteria inciting soft rot diseases;
(ii) to compare the general plant appearance and tuber production of the transgenic lines to non-transgenic control potato plants in the field; and
(iii) to perform tuber multiplication for future trials.
Statement for the Authority's public register
Application for approval under Section 40 (HSNO Act 1996) to field test, in Canterbury over 5 years, potato cultivars genetically modified for increased resistance to bacterial soft rots, to evaluate resistance and yield performance of individual lines.
10. Information on any likely inseparable organisms
The transgenic lines to be field tested have all been developed using high-health potatoes cultivars known to be free of all other organisms, including known diseases and pests.
Assessment of effects
11. Information on all the possible adverse effects of the organism on the environment 12. Identification and assessment of risks, costs and benefits and other impacts
13. Information on the positive effects of the organism 14. Assessment of effects
The ultimate outcome from this proposed field trial is the identification of potato lines with increased resistance to soft rot diseases that are suitable for further commercial development.
We believe the potential benefits of this proposed trial outweigh any potential costs.
Potential Risks (re; sections 11 and 12)
All the transgenic potato lines to be field tested contain two new genes: a kanamycin resistance marker gene and a gene anticipated to confer improved disease resistance. As outlined below, there is no information to suggest any consequences of the proposed small scale field trial of transgenic potato plants on tangata whenua, human or animal health, on target and non-target organisms, or on the general ecology, environmental quality and pollution in the area.
Furthermore, we can not envisage any such possible effects of the transgenic potato plants. The only possible deleterious effects of the introduced genetic trait that we can envisage are pleiotropic effects on the plant phenotype and the yield of tubers. The proposed field trial will examine these possibilities, in addition to field resistance to the appropriate disease. The only other possible risk is that of gene transfer to non-modified organisms. However, as outlined below and with the containment features in place (section 15), there are no organisms at the release site or in the surrounding environment to which possible transfer events can be envisaged.
Effects on public health and safety
From a scientific perspective, no adverse effects on human health and safety of these transgenic lines can be envisaged from the proposed small scale field trials. In contrast, some important benefit effects should result from the agricultural use of these transgenic potatoes (see below).
Effects on Maori culture and taonga
It is not expected that the proposed release would have any effect on Maori traditional resources.
The potato is an introduced crop plant and the plants which have been genetically modified and whose release is being sought represent cultivars already grown and/or bred in New Zealand.
We are at present consulting with iwi in our area over possible effects of the release of these genetically modified organisms. Iwi are being contacted through the iwi representative on the New Zealand Institute for Crop & Food Research Bioethics Committee. The representative is Mrs Cath Brown, nominated representative of the Ngai Tahu Trust Board, who will consult formerly with iwi over possible effects of the proposed small scale field trial of transgenic potatoes. Any concerns will be sent as a submission directly to ERMA. During consultations for previous field trials, approved by the IAG, on other transgenic potato lines modified with the same genes, the local Iwi reported that they did not have any problems with the applications (Appendix 1).
Effects on the environment, genetic diversity or significant deterioration of natural habitats
From a scientific perspective, no significant adverse effects on the New Zealand environment, or any significant deterioration of natural habitats in New Zealand, can be envisaged from the small scale field tests of transgenic potatoes. With respect to inherent genetic diversity in New Zealand, the consequences of these transgenic lines will be no different from that posed by the release of new potato cultivars developed by traditional breeding (Conner et al. 1997b). The gene transfer associated with the development of the transgenic potato lines represents an increase in genetic diversity within potato germplasm.
In contrast, the agricultural use of these transgenic potatoes should contribute to environmental enhancement and sustainability by reducing pesticide residues in soils and ground water. In respect to effects on natural habitats, the consequences of these transgenic lines will be no different from that posed by existing potato crops (Conner et al.
1997b); the transgenic potatoes will play the same role as existing potatoes (and most other higher plants) toward biogeochemical or biological cycling processes.
Affinities of the transgenic potatoes to other organisms
Potatoes in New Zealand are hosts for a wide range of viroid, viral, bacterial and fungal diseases, as well as and invertebrate pests such as insects and nematodes. In some instances potatoes are the only host to specific races of these pests and diseases. Previous research suggests the risk of gene transfer from the transgenic potatoes to associated pathogens is negligible (Schluter et al. 1995, Smalla et al. 1994).
It is also highly unlikely these transgenic potatoes will cause disease, be parasitic, or become a vector or reservoir for human, plant or animal disease as the transgenic potatoes lines are expected to behave in an identical manner to other potato crops, except for the improved resistance to specific pests or diseases.
It is highly unlikely that the transgenic potatoes will be consumed by larger animals such as birds or rodents. Potato berries (see below) contain alkaloids and, consequently, a bitter taste and neither birds or rodents are known to consume them. Although it is conceivable that rodents may dig up tubers, it is highly unlikely in such a small scale trial in an area with considerable human activity. This activity has never been observed in any of our previous transgenic potato trials over the past decade, so no provisions for deterrence of rodents is considered necessary.
There is unlikely to be a significant displacement or reduction in any native or other valued species as these transgenic potatoes have been specifically developed for improved resistance to soft rot diseases where the effects will be largely confined to individuals infecting the transgenic plants. The peptides being expressed have general activities (Jaynes et al. 1987, Zasloff et al. 1988; Wilson and Conner 1995) so the effects may extend to other microbial diseases and possibly even mycorrhizal fungi. However, any such effects are anticipated to be minimal compared to the current practice of applying broad spectrum pesticides to control potato diseases. The effective control of such diseases is the motivation for the current application for small-scale field contained trials.
Ability of transgenic potatoes to form undesirable self-sustaining populations, either by itself or following hybridisation with other organisms
In an agricultural context potatoes often appear as weeds in the following crops. These may arise from true seeds or from tubers remaining in the ground after harvest. The number of true seeds produced in fertile cultivars is highly dependent upon the cultivar grown, environmental conditions, and insect activity, but can be as high as 150-250 million per hectare (Accatino 1980;
Lawson 1983). Seeds may remain dormant in soil for up to 2 years and have been reported to retain viability over a seven year rotation to the next potato crop. Consequently, true potato seeds have the potential to become a weed problem in subsequent crops (Lawson 1983).
Potato plants as weeds can also arise from the inability to remove all tubers from a field during harvest, resulting in the appearance of volunteer (ground keeper or self-set) plants the following year. Up to 367,000 tubers per hectare have been estimated to remain in the field after mechanical harvest (Lutman 1977). Depending of the severity of the winter, many of these tubers will remain viable until the next spring. Potato tubers require 50 frost-hour equivalents at -2oC or below to kill tubers, eg 25 h at -2oC, or 5 h at -10oC etc (Lumkes 1974). In the temperate British climate, up to 80% of the tubers left after harvest died in even mild winters (Lutman 1977). The remaining viable volunteer tubers do not remain dormant and will always sprout the next season.
In addition to competing with the subsequent crop, the appearance of volunteer potatoes from
either true seed or tubers can carry through disease inocula that can affect the health of following potato crops. The survival of true seed in soil for many years may cause problems with maintaining genetic purity during the production of seed tubers.
Although volunteer potatoes can contribute to some problems in cropping situations, they are very limited outside cultivated crops. Potatoes are occasionally observed as small naturalised populations in New Zealand (Webb et al. 1988), usually on waste areas where household refuse has been dumped. They usually exist as transient populations and are not noted for forming long-term self-sustaining populations.
Twenty other species of Solanum are recognised as being naturalised in New Zealand. These species are: S. carolinense, S. chenopodioides, S. crispum, S. diflorum, S. dulcamara, S. jasminoides, S.
linnaeanum, S. marginatum, S. mauritianum, S. nigrum, S. physalifolium, S. pseudocapsicum, S. rantonnei, S.
rostratum, S. sisymbriifolium, S. villosum, S. americanum, S. aviculare, and S. laciniatum (Webb et al. 1988).
The latter three species are considered as native to New Zealand, but are also found in Australasia, and/or around the Pacific Ocean. S. crispum and S. jasminoides are also often grown as ornamental garden species. More recently naturalised populations of Solanum furcatum have been discovered near Kaikoura (A. J. Conner, unpublished observations). In addition to these species S. melongena (eggplant or aubergine) and S. muricatum (pepino) are commonly grown as edible crop plants. Other Solanum species are sometimes grown as ornamental plants in home gardens or as indoor plants, but have not become naturalised (yet). Of these species, S. nigrum (black nightshade) and S. physalifolium (hairy nightshade) are likely to occur as weeds in potato crops. Relative to potato, all of these species occur in a different Subgenus or different Section of the Solanum genus.
None of these species have been recorded to hybridise naturally with potatoes. This includes numerous attempts at crossing potatoes with New Zealand populations of many of these species, especially S. nigrum and S. physalifolium (see Table 1). Sterile F1 hybrids have been reported recently between potato and black nightshade, but only after resorting to very early embryo rescue and in vitro culture of the embryos (Eijlander and Stiekema 1994). The resulting interspecific hybrids were very weak and difficult to keep alive. Similar attempts to make this S.
tuberosum x S. nigrum cross elsewhere have failed (Dale 1993). Other attempts to hybridise S.
dulcamara and S. tuberosum have also failed (Eijlander and Stiekema 1994; Dale 1993). Gene transfer from transgenic potatoes to black nightshade was monitored during one of our earlier contained field trials in the 1989/1990 season. No evidence for gene transfer was obtained, despite screening over 53,000 black nightshade seedlings (Conner 1993, 1995). Similar results using 7,600 S. nigrum seedlings have been observed in Australia (Conner 1994), and well as for S.
nigrum and S. dulcamara in the United Kingdom (McPartlan and Dale 1994).
The opportunity for forming self-sustaining populations from the transgenic potatoes, or their sexual progeny following self pollination or hybridisation with other potatoes, is no different from than for existing potatoes (Conner et al. 1997b). If such populations did develop, the consequences would only be of nuisance value to the same extent as any other volunteer potato plant. In this context they would not considered as undesirable. The opportunity for undesirable self-sustaining populations forming following hybridisation with other Solanum species is minuscule given the failure of attempted hybridisation between these species.
Eradication of transgenic potatoes if they established an undesirable self-sustaining populations
In the highly remote event of these transgenic potato lines establishing an undesirable self-sustaining populations in New Zealand, it is highly unlikely that such populations will be even recognised as being derived from transgenic lines. If appropriate, such populations could be effectively eliminated by any broad spectrum herbicide that available for control of volunteer potatoes (e.g. amitrole, glyphosate, MCPA).
Other unlikely impacts
Additional unlikely impacts that have been proposed for transgenic plants include:
• the transgenic plants will become weeds;
• disease-resistant transgenic plants will cause ecological imbalance;
• growing disease-resistant transgenic plants will lead to the development of superpests;
• the transgenes will be transferred to weeds and enhance their weedy status; and
• the use of antibiotic-resistant markers genes may contribute to the development of antibiotic resistance in microbes.
However, traditional plant breeding, conventional agricultural practices, and existing situations in nature, which have been considered acceptable for many years, have similar risks to genetic engineering. More specifically, the risks of disease-resistant transgenic potatoes to natural ecosystems, agricultural ecosystems, food industries, and consumers will be no different than the effects of growing, processing and eating new cultivars with disease resistance developed via traditional potato breeding. A full analysis of these arguments can be found elsewhere (Conner 1997a, 1997b; Conner et al. 1997b).
There are no data to show that the incorporation of the kanamycin resistance gene or the pest and/or disease resistance genes into potatoes has any deleterious effect in the long term. Many thousands of field trials have been performed throughout the world over the last decade.
The majority of these field trials have involved transgenic plants with kanamycin resistance genes incorporated into their genomes. No deleterious effects have been reported in association with any of these field trials, apart from changes in the vigour of the transgenic plants themselves.
Potential Benefits (re; sections 12 and 13)
The development of transgenic potato cultivars with improved pest and disease resistance has benefits to all components of the food chain in the New Zealand potato industry, from the potato seed producers to the consumer. Potato seed producers and potato growers will benefit from the resistance to soft rot bacteria and other pests and diseases.
Elimination of the threat of the pests and diseases will result in more efficient growing of potatoes by substantially reducing the need for grading-out damaged tubers and increasing the yield of marketable tubers. It will also reduce or eliminate the need for applications of pesticides that are commonly used to reduce these disease problems, thereby making farming practices more efficient and economic. This will also contribute to environmental enhancement and sustainability by reducing pesticide residues in soils and ground water.
Potato processors will benefit from the more reliable supply of high quality tubers without soft rot damage. The reliable supply of high quality tubers to processors should result in an economic benefit for consumers. Consumers will also benefit from the reliable production of high quality table tubers without insect damage and with minimal pesticide residues and the consequent health benefits.
In a broader view, through the Foresight Project, an initiative run by the Ministry of Research, Science & Technology (refer www.morst.govt.nz/foresight for details), the New Zealand government has identified the need for new knowledge and technological innovation in order for NZ to remain competitive in the global economy. The rapidly expanding area of biotechnology is one of these new technologies and this proposed field trial will contribute to New Zealand’s development in this area.
15. Information on the proposed containment system
The trial will be planted and supervised by experienced staff from the Potato Breeding Section and the Plant Biotechnology laboratory of the New Zealand Institute for Crop & Food Research, Lincoln. It will be a standard potato field trial involving the planting of tissue cultured transplants and/or tubers produced in the containment greenhouses at Lincoln. This planting material will be produced in contained laboratories or greenhouses following protocols previously described (Conner et al. 1994). Micropropagated potato plants will be multiplied and maintain in vitro until 4-5 weeks prior to field planting, at which stage they will be transferred to the containment greenhouse for establishment in soil and gradual hardening-off for field transplantation. Tubers harvested from the containment greenhouse will be stored at 8oC within the containment facility for at least 6-8 weeks to fulfil any dormancy requirements. The exact number of required tubers or transplants will be transported to the trial site in secure boxes.
The genetically modified potatoes could theoretically disperse from the site as pollen, tubers or seeds. This proposed field trial and that of genetically modified potatoes with increased resistance to potato tuber moth are to be run side by side. Both experimental plots will be completely surrounded by 3 buffer rows of non-transgenic potato. This is essential for any agronomic trial to prevent "edge effects" and will also act as a buffer for pollen dispersal. These buffer rows will be planted with a potato genotype having purple tubers, to allow them to be readily distinguished from the white tubers of the transgenic potato lines. This will assist during the harvest of the proposed trial and subsequent monitoring of the trial sites.
To further minimise the risk of dispersal of the transgenic potatoes, the two trials will be isolated by at least 50 m from other non-transgenic potato trials to virtually eliminate any possibility of pollen transfer. Physical barriers are not considered necessary to prevent dispersal of these genetically modified potatoes, as an isolation distance of 20 m is considered to be the accepted international standard for isolation of transgenic potato field trials (Conner and Dale 1996).
The trial site is 100m from a few residential properties of Lincoln (Figure 2). Potatoes will be grown by the New Zealand Institute for Crop & Food Research in greenhouses 70 m from the proposed field trial (Figure 3). The majority of these will be located in "contained quarantine"
facilities associated with the Pathogen Tested Potato Scheme or the GMO containment greenhouse. Routine operations in each of these facilities involve restricted access, monitoring of insect screens over all vents and regular insecticide spraying programmes. This should prevent pollen transfer to these plants. A few potatoes will also be grown in other greenhouses for artificial pollination associated with the potato breeding programme at the New Zealand Institute for Crop & Food Research. It is anticipated that all of these plants will complete
flowering well before the plants of the proposed field trial begin flowering.
It is exceedingly unlikely that effective pollination will occur over these distances to neighbouring potato plants, since the monitoring of pollen dispersal from our previous field trials of transgenic potatoes established negligible pollen dispersal (Tynan et al. 1990; Conner 1993). Seeds harvested from wild-type plants growing at varying distances completely surrounding the field trials were screened for the genes inserted into the transgenic lines. This established that no effective pollination by transgenic pollen occurred at distances greater than 6.0 m from the field trial (despite screening of over 80,000 seedlings arising from pollination events 6 - 10 m from the trials). A Swedish study has suggested a 30% frequency for the presence of transgene-containing progeny from non-transgenic potato plants growing 1 km from a field trial of transgenic potatoes (Skogsmyr 1994). However, this study has been totally refuted on technical grounds (Conner and Dale 1996).
At harvest, the buffer rows of non-transgenic potato plants surrounding the field trial will be harvested via a mechanical lifter and hand picking. The plants in the experimental plots will be individually hand dug and picked. All berries developing on the transformed plants will be harvested and either be autoclaved or the seeds will be extracted for laboratory studies. After tubers have been dug, they will all be transported to the containment greenhouse facility [Plant House level one category, operated as recommended by the Advisory Committee on Novel Genetic Techniques (1994)] in secure sacks for counting and weighing of tubers. Bioassay tests for resistance against soft rot bacteria will be performed within the containment biotechnology laboratories [which have operated at category zero containment conditions since 1987 as recommended by the Advisory Committee on Novel Genetic Techniques (1994)].
Following harvest, the site will be grubbed several times with surface picking of remaining tubers. We do not anticipate any detrimental effects on the environment if the genetically modified potato plants remain in the field beyond the normal field trial period. If tubers remained in the ground after harvest they would become volunteers in the following crop and be only of nuisance value to the same extent as any other volunteer potato plant. Tuber dispersal would require human assistance. To prevent this, all harvesting and cultivation equipment will be thoroughly cleaned before use elsewhere, and all sacks containing harvested tubers will be securely tied closed before transport from the trial site.
All potato material used in bioassays will be disposed of via autoclaving. All other excess tubers will be disposed of via either autoclaving (for small amounts) or following the "drowning and burial" procedure outlined in section 8. The burial and trial sites will be monitored for at least two years following field testing for the appearance of volunteer potato plants which will be removed and destroyed. In the event of tubers remaining and growing the following season, the plants will be removed by hand digging.
Potato plants are very distinctive and can easily be recognised from other crop and weed species.
There is also a wide range of broad spectrum herbicides available for potato elimination (e.g.
amitrole, glyphosate, MCPA). Monitoring of our previous field trial sites for transgenic potatoes suggests that 1 - 3 volunteers/m2 can be expected the following season, which reduced to 0.01 - 0.2 volunteers/m2 in year 2, with none being observed in year three (Conner 1993; 1995).
Similar results would be expected using the harvesting, monitoring, and elimination procedures proposed for this field trial.
The containment procedures outlined above have proved very effective for previous field trials on transgenic potatoes over the past decade at Crop & Food Research, Lincoln.
16. Information on all occasions where the applicant is aware that the organism has been previously considered by a country or organisation and the results of such consideration All the potato lines to be assessed in the proposed field trials were developed in the plant biotechnology laboratories at Crop & Food Research, Lincoln. They have not been considered for field testing by any other country or organisation. There have been other field trials approved for transgenic potatoes throughout the world since 1986 (Dale 1995). Many of these have involved lines transformed with similar vectors to those described in this application. For example, there have been field trials on potato lines transgenic for cecropin B genes in North America (Belknap et al. 1994) and Europe (Allefs et al. 1995) and lysozyme genes in Germany (Klaus Düring, Institute for Breeding Methods in Vegetables, Quedlingberg, Germany, pers.
comm.).
17. Information on New Zealand's international obligations that may be relevant to the application
As far as the applicant is aware, there are no international obligations relevant to this application that may either deny or enforce the approval or rejection the application. All transgenic potato plants to be field tested were developed at Crop
& Food Research in Lincoln, and all the vectors used to develop these plants were developed in New Zealand.
18. Previous considerations
The development of the vectors used in this study and the transgenic potatoes were approved by the Advisory Committee on Novel Genetic Techniques (ACNGT). Field trials on transgenic potatoes containing many of the DNA sequences included in this application have been approved by the IAG over the past decade and the trials have been successfully performed and contained. These sequences include: Agrobacterium T-DNA derived border regions, promoter regions and terminator regions, the cauliflower mosaic 35S promoter, the kanamycin resistance selectable marker gene, the lysozyme gene from bacteriophage T4, and the artificial genes encoding the cecropin B analogue and magainin II. Some of the lines successfully evaluated in contained field trials (approved by the IAG) during the 1997/98 summer will be included in the proposed field trials. This is necessary to scientifically validate cross referencing of results between the different seasons.
19. Other relevant legislation
As far as the applicant is aware, there is no other legislation relevant to this application.
20. Glossary
35S Promoter -- a very active promoter of transcription isolated from a DNA plant virus called cauliflower mosaic virus
5’ end -- the “start” end of a mRNA or transcript
Agrobacterium tumefaciens -- a bacterium which causes crown gall disease, and has been exploited to genetically engineer plants because of its natural ability to transfer DNA into the plant cell nucleus
AMV leader sequence -- a small (38 base pair) sequence which increases the amount of protein made from a mRNA transcript when incorporated at the “start” of a transcript
autotetraploid -- a cell, tissue or organism with the presence of two chromosome sets of the same species
bacteriophage -- a virus whose host is a bacterial cell
BamH1 -- a restriction enzyme cleaving DNA at GGATCC sequences
binary vector -- a plasmid designed for Agrobacterium tumefaciens transfer of cloned genes into plant cells. It is called a binary vector because it must reside in the Agrobacterium tumefaciens cell with another plasmid for transfer to take place.
callus -- a tissue made up of undifferentiated cells
cecropin B -- a small peptide, consisting of 36 amino acids, from the giant silk moth with antibacterial activity
chimeric gene -- a gene comprising components from two or more other genes
codon -- a group of three adjacent nucleotides in an mRNA molecule that code either for a specific amino acid or for polypeptide chain termination during protein synthesis
codon usage -- the incidence of preferred codons for the specification of specific amino acids in polypeptides
EcoRI -- a restriction enzyme cleaving DNA at GAATTC sequences
"edge effects" -- the phenomenon of atypical plant performance in response to environmental effects as a consequence of plants growing at the edge of an agronomic trial
genome -- the inherited genetic material of an organism
HindIII -- a restriction enzyme cleaving DNA at AAGCTT sequences
heterozygous -- a genetic term which signifies that the copies of a gene present in an individual represent different versions (alleles) with the implication that the progeny of the individual will receive either or both versions of the gene
heterozygosity -- the condition of being heterozygous intercellular space -- the space between cells in a tissue kanamycin -- an antibiotic which can be lethal to plant cells
T4 lysozyme -- an enzyme from the bacteriophage T4 with specific hydrolytic activity against the bacterial cell wall component peptidoglycan (murein)
magainin II -- a small peptide, consisting of 22 amino acids, from the African clawed frog with antibacterial activity
micropropagation -- the clonal propagation of plants in tissue culture
mRNA -- an RNA molecule whose nucleotide sequence is translated into an amino acid sequence during polypeptide synthesis
neomycin phosphotransferase II -- the enzyme which modifies the antibiotic kanamycin so that it is no longer active
nopaline synthase gene -- a gene from naturally occurring Agrobacterium tumefaciens which is designed to function in a plant cell (not in a bacterial cell) and therefore carries signals useful for expressing genes in plant cells
northern analysis -- a method for detecting and characterising a specific RNA molecules in mixtures of RNA. Involves electrophoretic separation of RNA molecules by size, transferring these molecules to a membrane support, and detecting the specific sequence of interest by base pairing (hybridisation) to a known nucleic acid
NotI -- a restriction enzyme cleaving DNA at GCGGCCGC sequences nucleotide -- the basic unit structure of nucleic acids
octopine synthase gene -- a gene from naturally occurring Agrobacterium tumefaciens which is designed to function in a plant cell (not in a bacterial cell) and therefore carries signals useful for expressing genes in plant cells
pathogen -- a microorganism which causes disease phenotype -- the observed effect of a gene
plasmid -- a small circular chromosome found in bacterial cells which replicates autonomously from the bacterial genome
polyadenylation signal -- a part of a gene which specifies the addition of a tail of adenosine residues to the end of a mRNA, required for function of most genes in plant cells
polylinker -- a specially designed sequence of DNA used for cloning because it contains many different restriction enzyme cut sites in a row
polymerase chain reaction -- a biochemical reaction to synthesise many molecules of a specific DNA sequence
promoter -- a part of a gene which specifies the start of transcript (RNA) synthesis SacI -- a restriction enzyme cleaving DNA at GAGCTC sequences
signal peptide -- a region of a polypeptide chain that targets proteins to specific cellular compartments and is usually cleaved during transport across membranes.
Southern analysis -- a method for detecting and characterising a specific DNA sequence in a complex mixture of DNA sequences. Involves separating restriction enzyme digested DNA molecules by size, transferring these molecules to a membrane support, and detecting the specific sequence of interest by base pairing (hybridisation) to a known nucleic acid
T-DNA -- a piece of DNA which is transferred into plant cells by the bacterium Agrobacterium tumefaciens because the DNA contains transfer signals at its right and left borders
Ti-plasmid -- the naturally occurring plasmid in Agrobacterium tumefaciens which directs DNA transfer into plant cells
terminator -- see polyadenylation signal
tetrasomic -- a cell, tissue or organism with one chromosome represented four times
transcription -- the transfer of genetic information encoded in the nucleotide sequence of DNA into a nucleotide sequence of an RNA molecule
transgene -- a noun describing a gene which has been stably inserted into a cell or organism via a genetic engineering process
transgenic -- an adjective describing a cell or organism which contains added DNA introduced through a genetic engineering process
transformation -- the uptake of exogenous DNA by a cell and the recombination between that DNA and the DNA of the cell
translation -- the transfer of genetic information encoded in the nucleotide sequence of an
RNA molecule into the amino acid sequence of a polypeptide
transposon -- a transposable DNA sequence carrying one to many genes bound at each end by identical insertion sequences, which confer the ability to move from one location to another vir region -- a cluster of genes on the Ti-plasmid of Agrobacterium primarily responsible for the transfer and integration of T-DNA into plant cells
XbaI -- a restriction endonuclease cleaving DNA at TCTAGA sequences
21. Other relevant information
The field trial site is open to inspection by the ERMA (or an ERMA representative) before planting, while the potato crop is growing, or after harvest. Some scientific, industry, and public groups may be invited to visit the trial site; requests from other groups to visit the trial site may be granted, but will be at the discretion of Crop & Food Research.
The tubers harvested from the trial may be used for laboratory studies on biosafety assessment of transgenic potatoes. This may include evaluation of glycoalkaloid analyses and nutritional content.
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