NON-CONFIDENTIAL BUSINESS INFORMATION Directors: Ms. J. Janse Van Vuuren, Mr. L Matsiela (Non-Executive Director) Page | 1 Monsanto South Africa (Pty) Ltd Reg. No. 1968/001485/07 Monsanto House, Bldg No. 4 Fourways Office Park, Cnr. Roos & Fourways Boulevard, Fourways, 2055 P O Box 69933, Bryanston, 2021, South Africa Tel: (011) 790 8200, Fax: (011) 790 8442 VAT NO: 4440103507 02 June 2014 The Registrar Genetically Modified Organisms Act, 1997 Department of Agriculture, Forestry and Fisheries Directorate BioSafety Private Bag X 973 Pretoria, 0001 Dear Ms N. Mkhonza RE: Application for a time extension of an existing permit (39.4(4/13/254)) for activities with GMO’s in South Africa– Trial Release of MON 87460 at Malelane With this application Monsanto requests an extension of permit 39.4(4/13/254) authorizing Monsanto to produce a limited quantity of MON 87460 containing hybrid maize seed to be used for research purposes in South Africa and the WEMA partner countries. The quantity of MON 87460 containing hybrid seed produced during the seed make-up proposed in this application will be for research purposes only and will not be used for commercial use. The necessary quantities of seed needed for field trials in WEMA partner countries in sub-Saharan Africa will be exported to the respective countries at the required time. The MON 87460 containing hybrids that will be produced and the lines increased during the proposed trials will originate from inbred lines from the USA as well as inbred lines stored at the registered facility at Malelane (Registration no. 39.2/Monsanto SA-12/090). Additionally, the normal permit conditions do not allow us to utilize the exact same piece of land as the previous year, thus we also request the EC to maintain the amendment to Condition 30 as granted on 03-09-2013, reference 39/R, of the field trial conditions should our extension application be successful. Monsanto additionally request that it be allowed to enter the MON 87460 entries into the same trial as the entries containing MON 87460 x MON810, MON 87460 x MON 89034,
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NON-CONFIDENTIAL BUSINESS INFORMATION
Directors: Ms. J. Janse Van Vuuren, Mr. L Matsiela (Non-Executive Director)
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Monsanto South Africa (Pty) Ltd Reg. No. 1968/001485/07 Monsanto House, Bldg No. 4 Fourways Office Park, Cnr. Roos & Fourways Boulevard, Fourways, 2055 P O Box 69933, Bryanston, 2021, South Africa Tel: (011) 790 8200, Fax: (011) 790 8442 VAT NO: 4440103507
02 June 2014 The Registrar Genetically Modified Organisms Act, 1997 Department of Agriculture, Forestry and Fisheries Directorate BioSafety Private Bag X 973 Pretoria, 0001 Dear Ms N. Mkhonza RE: Application for a time extension of an existing permit (39.4(4/13/254)) for activities with GMO’s in South Africa– Trial Release of MON 87460 at Malelane With this application Monsanto requests an extension of permit 39.4(4/13/254) authorizing Monsanto to produce a limited quantity of MON 87460 containing hybrid maize seed to be used for research purposes in South Africa and the WEMA partner countries. The quantity of MON 87460 containing hybrid seed produced during the seed make-up proposed in this application will be for research purposes only and will not be used for commercial use. The necessary quantities of seed needed for field trials in WEMA partner countries in sub-Saharan Africa will be exported to the respective countries at the required time. The MON 87460 containing hybrids that will be produced and the lines increased during the proposed trials will originate from inbred lines from the USA as well as inbred lines stored at the registered facility at Malelane (Registration no. 39.2/Monsanto SA-12/090). Additionally, the normal permit conditions do not allow us to utilize the exact same piece of land as the previous year, thus we also request the EC to maintain the amendment to Condition 30 as granted on 03-09-2013, reference 39/R, of the field trial conditions should our extension application be successful. Monsanto additionally request that it be allowed to enter the MON 87460 entries into the same trial as the entries containing MON 87460 x MON810, MON 87460 x MON 89034,
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MON 87460 x MON 89034 x NK603 and MON 87460 x NK603. The trial will be used for efficacy evaluation and hybrid production of the mentioned traits and thus the trial will contain all these entries as well as commercial check varieties to facilitate this evaluation. These applications will be submitted separately from this extension application. The whole site will thus be treated as regulated. We would sincerely appreciate a review of this application. Please feel free to contact our office at any stage should additional information be required.
Yours faithfully J. Magson Regulatory Affairs Manager Monsanto South Africa
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APPLICATION FOR A TIME EXTENSION OF AN EXISTING PERMIT FOR
ACTIVITIES WITH GMO’S IN SOUTH AFRICA – TRIAL RELEASE
PART I: Please provide the following information: 1) Applicant / Party of Import
Monsanto S.A. (Pty) Ltd
2) Contact person [CBI-DELETED: Section 68(a),(b) and (c)ii of the Promotion of Access to Information Act]
3) Complete address Monsanto S.A. (Pty) Ltd Monsanto House, Building No.4, Fourways Office Park, Corner of Roos Street & Fourways Boulevard Fourways P.O.Box 69933 Bryanston 2021
4) Previous authorisations (permit number) and a detailed report of the activities conducted under the most recent permit. Approval to conduct production of MON87460 containing hybrid seed for research purposes was granted in 2013 under permit, 39.4(4/13/254). A trial report accompanies this application (Attachment A, [CBI-DELETED: Section
68(a),(b) and (c)ii of the Promotion of Access to Information Act]) reporting on trial
activities to date.
5) Characteristics of the GMO
Designation of the transformed line The developmental code is MON 87460
Phenotype
DIRECTORATE BIOSAFETY Private Bag X973, Pretoria, 0001
Harvest House Room 422, 30 Hamilton Street, Arcadia, Pretoria, 0002
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Except for the introduced trait, MON 87460 is equivalent to conventional maize.
Construct The functional genetic elements, location, origin and function of the inserted DNA in the MON 87460 plasmid construct PV-ZMAP595 are presented in Table 1 below. Construct PV-ZMAP595 encodes for the cold shock protein B gene from Bacillus subtilis. The plasmid map is provided in Figure 1 below. Any sections of the vector not listed in Table 1 but shown in the plasmid map, are non-transcribed vector sequences that do not contain any functional genetic elements. The plasmid used to create MON 87460 contains two expression cassettes. The first expression cassette produces CspB and the second produces neomycin phosphotransferase II (NPTII), a selectable marker that confers tolerance to aminoglycoside antibiotics such as neomycin. MON 87460 contains a single insert with the intended sequence and the insert is stably maintained over multiple generations according to the Mendelian laws of genetics.
Table 1: Genetic elements for PV-ZMAP595
Genetic Element
Localization (bp)
Size (bp)
Donor Function in the construct
Left Border 1 – 442 442 Agrobacterium tumefaciens
Octopine Left border sequence, essential for transfer of T-DNA.
Right border 5232-5588 357 Agrobacterium tumefaciens
Nopaline Right border sequence essential for T-DNA transfer
Act1 promoter
5621-6464 843 Oryza sativa The promoter from the actin 1 gene from Oryza sativa.
Act1 leader
6465-6544 80 Oryza sativa The 5’ untranslated region of the actin 1 gene from Oryza sativa.
Act1 intron 6545-7021 477 Oryza sativa The first intron from the actin 1 gene from Oryza sativa.
CspB 7024-7227 204 Bacillus subtilis The coding region of the Bacillus subtilis cold shock protein B gene which is believed to be involved in drought response.
Tr7 terminator
7258-7765 508 Agrobacterium tumifaciens
The 3' untranslated region of the transcript 7 (TR7) gene (which directs polyadenylation of mRNA).
Lox site 7840-7873 34 Bacteriophage P1 Recognition site for Cre recombinase from Bacteriophage P1.
The promoter for 35s transcript RNA derived from Cauliflower mosaic virus
NPTII 8257-9051 795 E. coli The neomycin phosphotransferase II gene (NPTII; confers resistance to kanamycin/neomycin) from E.coli
Nos terminator 9083-9335 253 Agrobacterium Tumefaciens
The 3’ nontranslated region from the nopaline synthase (NOS terminator) gene from Agrobacterium tumefaciens
Lox site 9361-9394 34 Bacteriophage P1 recognition site for Cre recombinase from Bacteriophage P1
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Figure 1: Plasmid map of PV-ZMAP595, used in MON87460
Promoter Act1 promoter: The fragment containing the rice actin 1 gene from Oryza sativa. It may be anticipated that a gene associated with the rice actin 1 promoter will have medium to high levels of constitutive expression, mainly in young tissues of roots, stem, leaves, meristems and seeds.
35S promoter The promoter for 35s transcript RNA derived from Cauliflower mosaic virus.
Gene CspB gene: Cold shock protein in construct PV-ZMAP595. The bacterial cold shock proteins bind to ssRNA and ssDNA (Lopez et al., 2001) and are thought to act as transcriptional anti-terminators by reducing secondary structure in bound RNAs (Bae et al., 2000) and/or slowing the degradation rate of polyadenylated messages (Feng, 2001). Some family members appear to play a role in response to changes in nutritional status in addition to cold stress resistance (Yamanaka and Inouye, 1997; 2001). Although the protein has been shown to be functionally redundant (Xia et al., 2001), differences in induction temperature (Wang et al., 1999) and binding specificity (Phadtare and Inouye, 1999) have been reported. CspB has been shown to confer
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chilling and freezing tolerance in transformed bacteria and has been shown to confer stress tolerance in transgenic rice.
Enhancer No enhancer regions were introduced in addition to the rice actin and 35S promoters.
Terminator (stop coding) Tr7 terminator: Transcription termination sequence: Tr7 3’ - The 3’ untranslated region of the transcript 7 gene from Agrobaterium tumefaciens, which directs polyadenylation of mRNA.
Selectable marker nptII: The neomycin phosphotransferase type II gene from TN5, a transposon isolated from Escherichia coli.
The gene nptII codes for neomycin phosphotransferase II and is used as a selectable marker. The bacterial enzyme confers resistance to antibiotics such as kanamycin, geneticin or neomycin and provides resistance by phosphorylating the antibiotics, rendering them inactive. Most plant species are sensitive to kanamycin and this provides a selective advantage for the growth of transformed plant cells.
6) Mode of transformation for each GMO MON 87460 was developed through Agrobacterium-mediated transformation of maize embryos and expresses cold shock protein B (CspB) from Bacillus subtilis. The transformation was performed with the binary plasmid vector PV-ZMAP595. The vector, PV-ZMAP595, contains both the left and right border sequences flanking the transfer DNA (T-DNA) to facilitate transformation. The Agrobacterium-mediated maize transformation to produce MON 87460 was based on the method described by Armstrong and Phillips (1988). Briefly, freshly isolated immature maize embryos were used for callus initiation. After co-culturing with Agrobacterium carrying the transformation vector, the calli were transferred from filter paper to callus initiation medium containing carbenicillin to eliminate Agrobacterium and paromomycin to eliminate cells that were not transformed, so that only cells containing the T-DNA survived. The resulting transformed cells were then subcultured several times on a selection medium and regenerated into plants. The R0 plants generated through the above transformation were self-pollinated, and the subsequent R1 plants were screened for the presence of CspB protein, tolerance to kanamycin, and homozygosity of the inserted gene. Only the plants that were homozygous for the cspB insert and tolerant to kanamycin were advanced for development, and their progenies were subjected to further molecular (Southern blot) and phenotypic assessments. MON 87460 was selected as the lead event based on its reduced yield loss under water-limited conditions, phenotypic characteristics, and molecular profile.
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Trait selection was performed using antibiotic resistance genes as markers. The gene nptII is of bacterial origin and codes for neomycin phosphotransferase II and is used as a selectable marker. The bacterial enzyme confers resistance to antibiotics such as kanamycin, geneticin or neomycin and provides resistance by phosphorylating the antibiotics, rendering them inactive. Most plant species are sensitive to kanamycin and this provides a selective advantage for the growth of transformed plant cells. The European Food Safety Authority has concluded that the use of antibiotic resistance markers has no adverse effects on human health and the environment (EFSA, 2009).
7) Aim of trial and estimated duration of the activity The purpose of the trial release is to produce hybrid seed containing the water-efficient maize event MON87460. The seeds will not be used for commercial purposes but solely for further research trials in South Africa and WEMA partner countries in sub-Saharan Africa. The goal of WEMA is to offer drought tolerant transgenic maize hybrids to small-holder farmers in Sub-Saharan Africa, royalty free, so that they are able to produce more reliable harvests. In this way, it is hoped that the project will contribute significantly to the improvement of food security, household income, livelihood and socio-economic development.
Our request to produce hybrid seed in South Africa has become necessary for a number of reasons. Firstly, it is critical for project success that the gene is tested in locally adapted germplasm and grown under local conditions. The trials conducted in South Africa, Kenya and Uganda to date has used mostly hybrids in a US germplasm or non-African adapted germplasm. Further benefits from producing the seed in South Africa would include a higher genetic purity as well as a reduction in the potential risk of adventitious presence. Seed produced at Malalane will be used for both local trials and exported to WEMA partner countries. The proposed trial release will take place on 1 ha at Monsanto’s breeding facility in Malelane. The prevention of pollen flow from any other maize to the trial is crucial for Monsanto, as purity of the seed harvested from this trial is essential in the hybrid development process. Hybrid seed production is strictly monitored in order to avoid contamination and measures to minimize the possibility of pollen flow to the trial will include border rows of conventional maize and appropriate time and distance isolation measures. Minimizing pollen flow to the trial will in return minimize pollen flow from the trial to any maize in the environment directly surrounding the trial location. A separate storage facility dedicated to the storage of the hybrid and parental line MON87460 seed will be used. The seeds produced will be dried, labeled and secured at this facility completely separate from any other maize seed. Following approval of the necessary export permits, seed will be transported to trial sites within South Africa or exported to approved trial locations in sub-Saharan Africa. Bearing in mind that precise handling and securing of the harvested seed is critical for Monsanto, adequate measures in terms of harvesting, bagging, transportation and storage of the seed from the trial site to the registered facility, would be taken. Accurate records would be maintained of all activities. These
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measures and records would also assist the monitoring activities by the Registrar’s office. Inspectors from the Registrar’s office would, as is a requirement in recent trial release permits, be informed in advance of the date of harvest to enable the inspectors to record the volumes harvested, seal the bags prior to transportation to the storage facility and again record the number of bags stored at the facility. We are confident that these measures will ensure effective containment of the harvested MON87460 containing seed. With this in mind, this request is made to the Executive Council for an extension of the hybrid make-up and parental line increase activities at Malelane. Our request is that a permit be issued to allow hybrid seed production and parental seed increase operations for 2014/2015.
8) Information pertaining to each trial site in terms of the following – (i) Ordnance survey map of appropriate scale with site marked
An aerial map is included in Attachment B [CBI-DELETED: Section
68(a),(b) and (c)ii of the Promotion of Access to Information Act].
(ii) GIS co-ordinates
The GIS co-ordinates of the sites are included in Attachment B [CBI-
DELETED: Section 68(a),(b) and (c)ii of the Promotion of Access to Information
Act].
(iii) Map indicating crops planted adjacent to the site and the distances involved Please see Attachment B [CBI-DELETED: Section 68(a),(b) and (c)ii of the
Promotion of Access to Information Act].
(iv) description in terms of –
volume seed to be planted (in kg) [CBI-DELETED: Section 68(a),(b) and (c)ii of the Promotion of Access to
Information Act]
size
soil
groundwater level
topography
flora and fauna
climate, especially prevailing winds
former use
distance from the nearest human settlements, along with the size of such settlements
distance from surface waters
distance from environmentally and otherwise protected areas
history of the site
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Details of the above are included in Attachment B [CBI-DELETED: Section
68(a),(b) and (c)ii of the Promotion of Access to Information Act].
(v) description of the environment immediately surrounding the release site; The environment surrounding the site is described in Attachment B [CBI-
DELETED: Section 68(a),(b) and (c)ii of the Promotion of Access to Information
Act]. (vi) the barriers planned in order to segregate the experiments
comprising the trial release from the surrounding environment; Monsanto is committed to effectively isolate the trials from any conventional maize growing in the environment surrounding the trial sites. Where other maize may be present in the environment immediately surrounding the trial sites, effective isolation measures will be implemented to prevent any cross-pollination between the trial site and other maize. Specific measures implemented at each site are provided in Attachment B [CBI-DELETED: Section 68(a),(b) and (c)ii of the Promotion of
Access to Information Act].
(vii) the supervision and monitoring of the trial release
Monsanto takes full responsibility for this activity and has assigned a dedicated person to actively manage and monitor the trial release. This dedicated person will be responsible for communicating any required information to the Registrar and attending all inspections by the authorities. These actions will be continuously monitored to determine progress made and to ensure compliance to the permit conditions. On completion of the trials, the trial site will be monitored for volunteer maize and any volunteers destroyed before flowering. If any unintended effect or environmental concern is raised, it will be reported within 48 hours, typically 12 hours, within Monsanto and further communicated to the Registrar, with the immediate implementation of appropriate measures to mitigate the circumstances or effect.
(viii) the contingency plans to deal with extreme conditions such as storms, floods and bush fires during the course of the trial release; Monsanto’s Personnel at the [CBI-DELETED: Section 68(a),(b) and (c)ii of
the Promotion of Access to Information Act] Research Station would monitor
the site. The station is located in an area that is mostly cultivated with sugarcane and occasionally peppers (on a small scale basis). The environment immediately surrounding the research station is therefore, to a large extent, also monitored through the day-to-day activities of the local farmers. This would result in a speedy reaction to/reporting of extreme conditions, such as bushfires. Furthermore, bushfires would be
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curbed by the river and stream as well as the fallow area and vegetation barrier around the site.
If any unintended effect or environmental concern is raised, it would be reported within 48 hours, typically 12 hours, within Monsanto and immediately further communicated to the Registrar, with the immediate implementation of appropriate measures to mitigate the circumstances or effect.
(ix) the provisions to remove or eliminate the GMO from the test site or any other place where it may be found upon completion of the trial release and to restore the test site and any such other place to its original form; After harvest, harvested grain will be secured and stored in a storage facility at the [CBI-DELETED: Section 68(a),(b) and (c)ii of the Promotion of
Access to Information Act] Breeding Facility. The remaining plant material
will be disked and ploughed. On completion of the trials, the trial site would be monitored for volunteer maize and any volunteers destroyed before flowering. As mentioned previously, being a trait introgression farm, strict control measures are in place.
(x) the arrangements for transporting the GMO to the release site. MON 87460 inbred seed will arrive at OR Tambo International Airport in sealed containers. The packaging will be clearly labelled to enable easy identification of the content. After clearing customs, the seed will be directly transported via courier service to Monsanto’s research facility near [CBI-DELETED: Section 68(a),(b) and (c)ii of the Promotion of Access to Information Act]. The seed will be secured and handled only at the trial location. After planting, the remaining parent seed will be securely stored. Records of the procedures described above will be maintained.
9) In the event of a contained use application; the type of facility, registration number of the facility and level of containment Not applicable.
10) Monitoring and accidents (i) Indicate the methods and plans for monitoring of the GMO (ii) Indicate any emergency procedures that will be applied in the event
of an accident Monsanto takes full responsibility for this trial and will assign a dedicated person to actively manage and monitor the trial at the site, who will be responsible for communicating any required information to the Registrar and attending all inspections by the authorities. These actions will be continuously monitored to determine progress made of the trial at each location and to ensure compliance to the permit conditions.
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On completion of the trial, the trial site would be monitored for volunteer maize and any volunteers removed before flowering.
If any unintended effect or environmental concern is raised, it would be reported within 48 hours, typically 12 hours, within Monsanto and further communicated to the Registrar, with the immediate implementation of appropriate measures to mitigate the possible impacts from the accident and work in consultation with the Registrar in terms of any other actions that may be required.
11) Pathogenic and ecological impacts (i) Submit an evaluation of the foreseeable impacts, in particular any
pathogenic and ecologically disruptive impacts. Seed destined for use in the hybrid seed production would be imported under the required permits and phytosanitary certificates. Use will also be made of remnant MON87460 parental seed stored at [CBI-DELETED:
Section 68(a),(b) and (c)ii of the Promotion of Access to Information Act] in a
registered facility. Monsanto has been conducting maize field trials in terms of the GMO Act for numerous years, with no ecological disruptive impacts recorded as a result of these trials. In addition, considering that there are no wild relatives of maize in SA and that maize, including MON 87460, requires human intervention to succeed, no ecological disruptive impacts are anticipated during these field trials. The containment conditions as stated previously have successfully allowed transgenic crops to be evaluated without any ecological impacts for the last ten years. We expect that this release will be the same as its forerunners.
12) Risk management (i) Please indicate any risk management measures that would be
required during the activity. Potential risks related to pollen flow from the trial sites to other maize would be managed effectively through implementation of appropriate reproductive isolation measures. On completion of the trial, any volunteers will be removed to address the potential risks associated with the dissemination of the event after the trial is completed. No further risk management measures are envisaged.
13) What are the implications of the proposed activity with regard to the health and safety of the workers, cleaning personnel and any other person, that will be directly or indirectly involved in the activity? Please take into consideration the provisions of the Occupational Health and Safety Act, 1993 (Act No. 181 of 1993) and accompanied regulations. Except for the introduced trait, MON 87460 is equivalent to conventional maize and would be handled in the same manner as conventional maize. No additional
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measures other than the normal measures applied during handling of conventional maize, would be required.
14) Indicate the proposed health and safety measures that would be applied to safeguard employees during the proposed activity. As was indicated above, MON 87460 requires no additional safety measures. However, Monsanto takes seriously the responsibility of the safety of all workers involved in Monsanto field trials. Monsanto’s safety standards, regardless of whether the seed is GM or not, are described below. Safe guarding of employees:
o Monsanto has a health and safety plan in place, is OSHAS certified and is regularly audited by National Quality Assurance.
o All employees using implements/equipment undergo regular training to ensure correct usage of all equipment. All employees are also informed of all hazards relating to the equipment.
o Job safety analysis has been drawn up for all equipment and is posted at various areas on site. Training on Job Safety Analysis is also done on an ongoing basis.
o All employees are issued with the required personal protective clothing and equipment to ensure that they are protected while operating all implements/equipment.
o Monsanto has a procedure in place for reporting of all incidents. Actions are then taken to implement preventative measures to prevent these types of incidents from re-occurring.
o All implements and equipment have been guarded to protect employees from exposure to chains, sprockets, gears etc.
o Noise surveillance is done on all equipment and employees undergo annual hearing tests.
o Employees undergo annual medical checks.
15) COMPLETE THE AFFIDAVIT. The completed affidavit is provided with this application [CBI-DELETED: Section
68(a),(b) and (c)ii of the Promotion of Access to Information Act].
16) COMPLETE THE RISK ASSESSMENT DOCUMENT (PART II). The completed Risk Assessment is provided below as Part II.
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REFERENCES Armstrong, C.L. and Phillips, R.L. 1988. Genetic and cytogenetic variation in plants regenerated from organogenic and friable, embryogenic tissue cultures of maize. Crop Science 28: 363-369 Bae, W., Xia, B., Inouye, M., and Severinov, K. 2000. Escherichia coli CspA-family RNA chaperones are transcription antiterminators. Proc. Natl. Acad. Sci. USA. 97: 7784-7789 EFSA. 2009. Consolidated presentation of the joint Scientific Opinion of the GMO and BIOHAZ Panels on the “Use of Antibiotic Resistance Genes as Marker Genes in Genetically Modified Plants” and the Scientific Opinion of the GMO Panel on “Consequences of the Opinion on the Use of Antibiotic Resistance Genes as Marker Genes in Genetically Modified Plants on Previous EFSA Assessments of Individual GM Plants”. http://www.efsa.europa.eu/EFSA/efsa_locale-1178620753812_1211902604575.htm Feng, Y., Huang, H., Liao, J., and Cohen, S.N. 2001. Escherichia coli poly(A)-binding proteins that interact with components of degradosomes or impede RNA decay mediated by polynucleotide phosphorylase and RNase. E. J. Biol. Chem. 276: 31651-31656 Lopez, M.M., Yutani, K., and Makhatadze, G.I. 2001. Interactions of the cold shock protein CspB from Bacillus subtilis with single-stranded DNA. Importance of the Tbase content and position within the template. J. Biol. Chem. 276: 15511-15518 Phadtare, S., Alsina, J., and Inouye, M. 1999. Cold-shock response and cold-shock proteins. Curr. Opin. Microbiol. 2: 175-180. Rissler, J. and Mellon, M. 1993. Perils amidst the Promise: Ecological risks of Transgenic Crops in a Global market. Union of Concerned Scientists, Cambridge, MA. Pp.22 Wang, N., Yamanaka, K., and Inouye, M. 1999. CspI, the ninth member of the CspA family of Escherichia coli, is induced upon cold shock. J. Bacteriol. 181: 1603-1609 Xia, B., Ke, H., Jiang, W., and Inouye, M. 2001. The Cold Box stem-loop proximal to the 5'-end of the Escherichia coli cspA gene stabilizes its mRNA at low temperature. J. Biol. Chem. 277: 6005-6011 Yamanaka, K., and Inouye, M. 1997. Growth-phasedependent expression of cspD, encoding a member of the CspA family in Escherichia coli. J. Bacteriol. 179: 5126-5130 Yamanaka, K., and Inouye, M. 2001. Induction of CspA, an E. coli major cold-shock protein, upon nutritional upshift at 37 degrees C. Genes Cells. 6: 279-290 These references are available should they be required.
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AFFIDAVIT/VERKLARING/STATEMENT
[CBI-DELETED: Section 68(a),(b) and (c)ii of the Promotion of Access to Information Act]
PART II
Risk Assessment (In accordance with Annex III of the Cartagena Protocol on Biosafety)
Risk assessment details
1. Country Taking Decision:
South Africa
2. Title: Jacques Magson Regulatory Affairs Manager
3. Contact details: Monsanto Company, represented by Monsanto S.A.(Pty) Ltd Monsanto Company 800 N. Lindbergh Boulevard . St. Louis, Missouri 63167 U.S.A Monsanto House, Building No. 4 Fourways Office Park Corner Fourways Boulevard and Roos Streets Fourways South Africa
LMO information
4. Name and identity of the living modified organism:
Maize lines containing MON 87460 event.
5. Unique identification of the living modified organism:
MON-8746Ø-4
6. Transformation event:
MON 87460.
7. Introduced or Modified Traits:
A. Abiotic environmental tolerance - Drought or water tolerance
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8. Techniques used for modification:
Agrobacterium-mediated transformation of embryonic maize cells.
9. Description of gene modification:
Maize was genetically modified to encode for the cold shock protein B (CspB) gene from Bacillus subtilis to produce maize plants that are tolerant to abiotic stress factors.
Characteristics of modification
10. Vector characteristics (Annex III.9(c)):
The plasmid vector PV ZMAP595 is derived from Bacillus subtilis, a soil bacterium ubiquitous in nature.
11. Insert or inserts (Annex III.9(d)):
The following inserts: MON 87460 is a transformant of plasmid vector PV-ZMAP595. Other regulatory inserts include Act1, a fragment containing the rice actin gene from Oryza sativa; transcriptional sequence Tr7 3’ the 3’ untranslated region of the transcript 7 gene from Agrobacterium tumefaciens that directs polyadenylation of mRNA; and cold shock protein B from Bacillus subtilis.
Recipient organism or parental organisms (Annex III.9(a)):
12. Taxonomic name/status of recipient organism or parental organisms:
Common name: Maize Family name: Gramineae Genus: Zea Species: mays(2n+20)
13. Common name of recipient organism or parental organisms:
Maize or Corn.
14. Point of collection or acquisition of recipient or parental organisms:
The original transformations that produced MON 87460 used privately owned germplasm acquired for this purpose.
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15. Characteristics of recipient organism or parental organisms related to biosafety:
Maize is the world’s third leading cereal, following rice and wheat, in terms of production and area harvested. It has a long history of safe use as a raw material for processed products, and direct uses as a human food or animal feed. Today, maize is produced on every continent except Antarctica, and is exported and imported as viable grain for use as foods or feeds, or directly in processing, without risk to the environment.
According to the OECD [Consensus Document on the Biology of Zea mays subsp. mays (Maize), 2003], “Maize has lost the ability to survive in the wild due to its long process of domestication, and needs human intervention to disseminate its seed.” In addition, “maize is incapable of sustained reproduction outside of domestic cultivation”, and “maize plants are non-invasive in natural habitats.” Despite the fact that maize frequently appears as a volunteer plant in a subsequent rotation, it has no inherent ability to persist or propagate. In all regions of the world, volunteer plants are managed with herbicides, tillage, or manual removal of plants. As such, maize is not considered to be a pest anywhere in the world. When it occurs outside of cultivation, it has no impact on the conservation and sustainable use of biological diversity.
Gene flow from maize occurs through dispersal of seed and pollen-mediated exchange of genes to sexually compatible plants. Since maize has no biological mechanism to scatter seed, low-level, incidental dispersal of viable grain occurs as a result of human-based activities such as transport and harvesting operations. As was noted by the OECD, the few plants that might result from incidental release will not persist or meaningfully reproduce without human intervention. Gene flow via pollen is only possible to other maize plants throughout the world except in Mexico and Guatemala where wild relatives occur. Maize reproduces sexually, is a wind-pollinated, monoecious species with separate staminate (tassels) and pistillate (silk) flowers, which encourages natural cross-pollination between maize plants. The distance that viable pollen can travel depends on prevailing wind patterns, humidity, and temperature. Generally, the pollen dissemination period lasts three to seven days. Because incidental release of maize during importation occurs at very low levels, and because maize in not competitive, pollen-mediated gene flow between local maize and rare volunteers has had no effect on the conservation and sustainable use of biological diversity.
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16. Centre(s) of origin of recipient organism or parental organisms:
Maize is thought to have its origin in Mexico, from where it spread northward to Canada and southward to Argentina. Although secondary centres of origin in South America are possible, the oldest archaeological evidence of domesticated maize (5000 B.C.) was discovered in the valley of Tehuacan in Mexico (Benson and Pearce, 1987). Several theories on the origin of maize have been proposed; the two theories most adhered to being that either teosinte (a wild relative of maize that is endemic to parts of Mexico and Guatemala) or a wild pod maize that is now extinct, was the wild ancestor of maize (Benson and Pearce, 1987; Brown et al., 1984).
Maize is a member of the genus Zea, which is broken into 2 sections: ZEA and LUXURIENTES. The section ZEA includes one species (mays), which includes three subspecies: ssp. mays, ssp. mexicana (formerly Euchlaena mexicana), and ssp. parviglumis. The former subspecies is known as maize, while the latter comprise a portion of the complex known as teosinte. Furthermore, ssp. mexicana and ssp. parviglumis are further separated into several races (OECD, 2003). Section LUXURIENTES encompasses 3 species: an annual Z. luxurians, and perennials Z. diploperennis and Z. perennis. While the classification of Zea continues to be modified, teosintes are the only know wild relatives of maize capable of forming hybrids in nature. Outcrossing and gene exchange between teosinte and maize has been reported with annual teosinte (Zea mays ssp. mexicana) (2n = 20) and maize (Zea mays L.) (2n = 20). A frequency of one F1 hybrid (maize × teosinte) for every 500 maize plants or 20 to 50 teosinte plants in the Chalco region of the Valley of Mexico was reported. However, newer information shows that annual teosintes may be a separate species because of the level of genetic isolation and that hybrids that do form are highly unsuccessful in introgressing genetic material (OECD, 2003). Regardless, Mexico and parts of Central America are regarded as the center of genetic diversity for maize. The natural distribution of teosinte is limited to the seasonally dry, subtropical zone with summer rain along the western escarpment of Mexico and Guatemala and the Central Plateau of Mexico.
The belief that Central America and southern Mexico are both the center of origin and a center of diversity for maize was supported by Vavilov (1992).
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17. Centres of genetic diversity, if known, of recipient organism or parental organisms:
See 16 above.
18. Habitats where the recipient organism or parental organisms may persist or proliferate:
As noted by the OECD (2003), maize is not invasive of natural habitats and does not persist or disperse anywhere in the world without human intervention. Early domestication and diversification through selection occurred in Meso-America. Maize is grown across a wide range of ecological conditions including soil types, altitude and rainfall. Currently, maize is grown over a wide range of conditions because of its many divergent types that have been bred for this purpose. Most maize is produced between latitudes 30° and 55°, with relatively little grown at latitudes higher than 47° latitude anywhere in the world. The greatest maize production occurs where the warmest month isotherms range between 21 and 27° C and the frost-free season lasts 120 to 180 days. A summer rainfall of 15 cm is approximately the lower limit for maize production without irrigation.
Experience with maize imported for use as foods or feeds, or directly in processing, has demonstrated that stable populations do not establish, persist or proliferate as a result of this practice.
Donor organism or organisms (Annex III.9(b)):
19. Taxonomic name/status of donor organism(s)
Bacillus subtilis
20. Common name of donor organism(s):
Soil bacterium
21. Point of collection or acquisition of donor organism(s):
Organisms are ubiquitous in nature and commonly found in soil.
22. Characteristics of donor organism(s) related to biosafety:
Not applicable, since the donor organisms are ubiquitous in nature and therefore do not pose a threat to biodiversity.
Intended use and receiving environment
23. Intended use of the LMO (Annex III 9(g)):
The objectives of the trial would be to assess the ability of MON 87460 to use water efficiently under normal, severe and catastrophic drought conditions.
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24. Receiving environment (Annex III.9(h)):
Trial location in Malalane
Risk assessment summary
25. Detection/Identification method of the LMO (Annex III.9(f)):
Standard molecular biology techniques may be used for detection including, but not limited to Southern or PCR, for terminators and promoters in this construct. A detection method for MON 87460 was provided to the Registrar in March 2009. The detection method for MON 87460 is considered trade secret or otherwise confidential information of Monsanto and is to be distributed by Monsanto only.
26. Evaluation of the likelihood of adverse effects (Annex III.8(b)):
Based on the nature of the recipient species (unable to proliferate) and the lack of related and wild species with which MON 87460 can outcross, the likelihood of adverse effects from out-crossing to other related species is negligible.
Transgenic maize hybrids with similar genes have been grown around the world for several years and in South Africa for 15 years without any recorded impact on the environment other than those created by conventional maize production.
Any volunteers could, like conventional maize, be removed by current agricultural practices such as ploughing and the use of herbicides.
27. Evaluation of the consequences (Annex III.8(c)):
Studies conducted with MON 87460 confirmed that this event is agronomically and compositionally equivalent to conventional maize and has no increased tendency towards weediness or an increased susceptibility of tolerance to insects normally associated with maize. Thus, should any of the potential risks materialize, the consequences would be negligible.
28. Overall risk (Annex III.8(d)):
Considering the potential risks and the consequences should the potential risks materialize, the overall risk of conducting field trials with MON 87460 at the selected site is extremely low.
29. Recommendation (Annex III.8(e)):
No risks have been identified and therefore other than the containment parameters that might apply through the permit conditions, no additional actions need to be taken.
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30. Actions to address uncertainty regarding the level of risk (Annex III.8(f)):
The potential risks identified for the specific sites are negligible, hence no additional actions are required except compliance with the conditions contained in the permit.
Additional information
31. Availability of detailed risk assessment information:
More information on the safety of MON 87460 is contained in the application preceding this document.
32. Any other relevant information:
None
33. Attach document: Not applicable to applicant
34. Notes:
References:
Benson, G.O. and Pearce, R.B. 1987. Corn perspective and culture. Corn: Chemistry and Technology, Chapter 1. Brown, W.L., Zuber, M.S., Darrah, L.L. and Glover, D.Y. 1984. Origin, adaptation, and types of corn. Corn: Chemistry and Technology. OECD. 2003. Consensus document on the biology of Zea mays subsp. mays (maize). Vavilov, N.I. 1992. Origin and geography of cultivated plants. These references are available should they be required.
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ATTACHMENT A
FINAL ANNUAL TRIAL REPORT
TRIAL RELEASE OF MON87460 DURING GROWING SEASON 2013 AT MALALANE
PERMIT NUMBER: 39.4(4/13/254)
[CBI-DELETED: Section 68(a),(b) and (c)ii of the Promotion of Access to Information Act]
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ATTACHMENT B
Trial Site Information and Trial Design
[CBI-DELETED: Section 68(a),(b) and (c)ii of the Promotion of Access to Information Act]
