NON-CONFIDENTIAL BUSINESS INFORMATION for a time extension of an existing... · expresses the Cspb...

NON-CONFIDENTIAL BUSINESS INFORMATION

Directors: Mr. W.A.J. Steenekamp, Mr. M.D. Ndlovu, Ms S.P. Ndebele, Mr. K.N. Pillay 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 8350 VAT NO: 4440103507

23 June 2015 Ms N Mkhonza Registrar: Genetically Modified Organisms Act, 1997 Directorate BioSafety Department of Agriculture Private Bag X973 Pretoria 0001

Re: Application for a Time Extension of an existing permit (39.4(4/14/398)) for activities with GMO’s in South Africa – Trial Release of MON 87460 x NK603 at Lutzville, Orania, Hopetown and Malelane Dear Ms Mkhonza Monsanto hereby requests authorization to continue field trials with MON 87460 x NK603 at four locations (Lutzville, Orania, Hopetown and Malelane) during the 2015-2016 growing season. Monsanto South Africa first obtained approval to conduct field trials with MON 87460 x MON 89034 x NK603 in 2014. Efficacy evaluation was conducted at Lutzville, Orania, Hopetown and Malelane. Reports for the previous seasons’ trials will be submitted the Registrar as soon as data analysis is completed; a preliminary report accompanies this extension application in the meantime. The main objective of these trials, as was the case in the current growing season, is to confirm the performance and efficacy of MON 87460 × NK603 maize under water-limited conditions and the tolerance to glyphosate. In addition to the previous point, Monsanto also request permission to conduct line increases and hybrid make-up at one of the mentioned sites, the Malelane site. This will allow Monsanto to produce hybrids and lines for further efficacy testing and efficacy evaluation. 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 of the MON 87460 single event should our extension application be successful.


Directors: Mr. W.A.J. Steenekamp, Mr. M.D. Ndlovu, Ms S.P. Ndebele, Mr. K.N. Pillay Page | 2

Monsanto also request that it be allowed to enter MON 87460 x NK603 entries into the same trial as the entries containing MON 87460 x MON810, MON 87460 x MON 89034, and MON 87460 x MON 89034 x NK603. The trial will be used for efficacy evaluation 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.

In view of the above, we kindly request approval to continue trials with MON 87460 x NK603 maize for a further growing season. We trust that this application will meet the requirements for assessment and that the application would be approved to enable the continuation of the field trials in the 2015-2016 growing season. Yours faithfully

DELETED: Section 68(a), (b) and (c) ii of the Promotion of Access to Information Act

MON 87460 x NK603 Extension Application

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 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 confined field trials with MON 87460 x NK603 was first granted in 2014, permit (39.4(4/14/398)), for activities with GMO’s in South Africa at Lutzville, Orania, Hopetown and Malelane. A preliminary report for the 2014-2015 season is provided in Attachment A (DELETED: Section 68(a), (b) and (c) ii of the Promotion of Access to Information Act).

5) Characteristics of the GMO

Designation of the transformed line The developmental code is MON 87460 x NK603

Phenotype

DIRECTORATE BIOSAFETY Private Bag X973, Pretoria, 0001

Harvest House Room 422, 30 Hamilton Street, Arcadia, Pretoria, 0002

Tel: 12 319 6382, Fax: 12 319 6329, E-mail: [email protected]



Except for the introduced trait, MON 87460 x NK603 is equivalent to conventional maize. The combination was produced via conventional crossing of lines containing the individual events. The stacked product thus expresses the Cspb and CP4 EPSPS proteins, resulting in MON 87460 x NK603 maize hybrids being tolerant to herbicides containing glyphosate, the combination is also more tolerant to drought stress.

Construct – MON 87460 MON 87460 × NK603 results form traditional breeding of MON 87460 and NK603. The inserted DNA fragments from both inbred parental lines are inherited in MON 87460 × NK603. Genetic modification was used in the development of MON 87460 and NK603. The DNA fragments present in MON 87460 × NK603 are inherited from MON 87460 and NK603. The individual components, location, source and function of these inherited sequences are given in Tables 1 and 2 (DELETED: Section 68(a), (b) and (c) ii of the Promotion of Access to Information Act).

For estimating the sizes of the bands present, the molecular weight markers on the left of each Southern blot were used.



Figure 1. Map of Plasmid Vector PV-ZMAP595

Circular map of the plasmid vector PV-ZMAP595 used to generate MON 87460. Genetic elements are depicted as arrows and are annotated on the exterior of the map. Restriction sites (with positions relative to the size of the plasmid vector) are shown for enzymes used in the Southern analysis on the exterior of the map. The restriction enzyme Sca I does not cut PV-ZMAP595. Probes used in the Southern analysis are shown as bold arcs on the interior of the map. Specific descriptions of the probes are given in the accompanying table.

Probe DNA Probe Start

Position End

Position Total Length

(~kb)

1 CS-cspB Probe 4608 4811 0.2 2 CS-nptII Probe 5839 6635 0.8

PV-ZMAP5959379 bp

Blp I 2140

EcoR V 5041

Msc I 6051

Msc I 7905

Msc I 8963

Ssp I 4935

Ssp I 5016

Ssp I 7618

Xba I 5376

CS-rop

OR-ori-pBR322

aadA

B-Right Border

P-Ract1

I-Ract1

CS-cspB

T-tr7

loxP

P-35S

CS-nptII

T-nos

loxP

B-Left Border

OR-ori V

2

1



Figure 2. Map of Plasmid Vector PV-ZMGT32

Circular map of the plasmid vector PV-ZMGT32 used to generate NK603. Genetic elements are depicted as arrows and are annotated on the exterior of the map. Restriction sites (with positions relative to the size of the plasmid vector) are shown for enzymes used in the Southern analysis on the exterior of the map. The probe used in the Southern analysis is shown as a bold arc on the interior of the map. A specific description of the probe is given in the accompanying table.

* These two fragments have the same nucleotide sequence and both represent probe 5.

Probe DNA Probe Start Position

End Position

Total Length (~kb)

5 TS-CTP2/ CS-cp4 epsps 1609 3204 1.6* 4959 6554 1.6*

9308 bp

P-Ract1

I-Ract1

TS-CTP2

CS-cp4 epsps

T-nos

P-e35S

I-Hsp70

TS-CTP2

I-Ract1

TS-CTP2

CS-cp4 epsps

T-nos

P-e35S

I-Hsp70

TS-CTP2

CS-cp4 epsps

OR-ori-pBR322

CS-nptII

PV-ZMGT32

Sca I 4704

Msc I 8742

5

5

T-nos

EcoR V 169

EcoR V 4010

EcoR V 6828

Ssp I 4682



Promoter Following is a summary of the promoter regions found in MON 87460 x MON 89034 x NK603 produced via conventional breeding. (DELETED: Section 68(a), (b) and (c) ii of the Promotion of Access to Information Act)

Gene The genome of MON 87460 x NK603 contains cold shock protein B (CSPB) inherited from MON 87460 as well as the NptII protein which functions as a selectable marker; it also contains the CP4 EPSPS proteins inherited from NK 603. Biological activity of these proteins is described below.

CspB protein

The CspB protein is an RNA chaperone which is associated with enhanced abiotic stress acclimation and tolerance is conferred through the destabilization of RNA secondary structure. CspB thereby facilitates RNA translation in B. subtilis (Phadtare et al., 2002a; Phadtare et al., 2002b). A codon optimized cspB coding sequence was used in the PV-ZMAP595 vector. The amino acid sequence of the CspB protein in MON 87460 coded by the PV-ZMAP595 vector, however, is identical to the native CspB protein (Willimsky et al., 1992) produced in Bacillus subtilis with the exception of one amino acid change in the second position from leucine to valine, designated as CspB-L2V. This amino acid change was implemented to facilitate the assembly of the plasmid vector PV-ZMAP595 for plant transformation.

NptII protein

The NptII protein functions as a selectable marker in the initial laboratory stages of plant cell selection following transformation (De Block et al., 1984; Horsch et al., 1984). The NptII enzyme uses ATP to phosphorylate neomycin and related aminoglycoside antibiotics, thereby inactivating them. Cells that produce the NptII enzyme selectively survive exposure to these aminoglycosides. The nptII coding sequence is derived from the prokaryotic E. coli transposon Tn5 (Beck et al., 1982). The purpose of inserting the gene encoding the NptII protein into maize cells along with CspB was to have an effective method for selecting cells after transformation. In general, the efficiency of plant cells transformation is often low, ranging from 1 × 10-5 to 1 × 10–4 of cells treated (Fraley et al., 1983). Therefore, the selectable marker, NptII, was used to facilitate the screening process.

CP4 EPSPS proteins

The CP4 EPSPS proteins provide tolerance to glyphosate (N- phosphonomethyl-glycine), the active ingredient in the non-selective, foliar-applied, broad-spectrum, post-emergent herbicide Roundup®1 herbicide formulations, resulting in excellent weed control with favourable environmental and safety characteristics. However, the sensitivity of crop plants to glyphosate has prevented the in-season use of this herbicide over-the-top of crops. The extension of the use of Roundup

1

® Roundup is a registered trademark of Monsanto Technologies LLC



herbicide to allow in-season application in major crops such as maize provides novel weed control options for farmers. The use of Roundup in maize is significant as it enables the farmer to take advantage of the herbicide’s favourable environmental properties.

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. T-nos Transcript termination sequence of the nopaline synthase (nos) coding sequence from Agrobacterium tumefaciens, which terminates transcription and directs polyadenylation

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.

In addition would the presence of the CP4 EPSPS protein enable selection through tolerance to herbicides containing glyphosate.

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. NK 603 was generated by particle acceleration technology using a linear DNA fragment PV-ZMGT32L.

7) Aim of trial and estimated duration of the activity The aim of the field trial release is to determine the performance and efficacy of the stacked maize product, MON 87460 × NK603, tolerance to water-limited conditions and tolerance to glyphosate. Information generated might be used in a future application for general release in South Africa, also might be used as supporting data for applications in other world regions.



Testing any abiotic stress tolerance transgene requires an in-depth knowledge of the field trial sites that can only be obtained over multiple years of trials in the same field. Experience in other world areas has shown that it is necessary to conduct trials over multiple years in order to obtain a robust data set. Monsanto has already conducted one season’s testing during 2014-2015 (Attachment A(DELETED: Section 68(a), (b) and (c) ii of the Promotion of Access to Information Act)), at least one more season of data is needing in order to generate enough local data to support a potential General Release application in South Africa.

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 (DELETED: Section 68(a), (b) and (c) ii of the Promotion of Access to Information Act).

(ii) GIS co-ordinates

The GPS co-ordinates of the sites are included in Attachment B (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 refer to Attachment B (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) In total, (DELETED: Section 68(a), (b) and (c) ii of the Promotion of Access to Information Act) kg of seed is required to perform the trials.

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 Details of the above are included in Attachment B (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 (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. Monsanto is committed to effectively segregate the trials from any other maize growing in the environment immediately surrounding the trial sites. Where other maize may be present in the fields immediately surrounding the trial sites, effective isolation measures such as 500 m distance isolation or a 4 week (+ 70m distance) temporal isolation, would be implemented to prevent any cross-pollination from the trial site.

(vii) The supervision and monitoring of the trial release Monsanto takes full responsibility for these trials and will assign a dedicated person to actively manage and monitor the trial at each location. Data pertaining to standard agronomic traits such as root lodging, stalk lodging, yield, grain moisture, plant height, stand count, barrenness and leaf firing will be captured during the trial. The dedicated person at each trial site 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. On completion of the trials, the trial sites 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; The environment immediately surrounding the trial sites includes soya beans, natural veld and maize. Therefore, to a large extent, the environment directly surrounding the trial sites is continuously monitored through day-to-day activities by the local farmers and/or residents, which



would result in a speedy reaction to/reporting of any incidents such as bushfires.

The potential risk related to flooding of the sites is very low. If any unintended effect or environmental concern is raised, it will 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 collecting the necessary data, harvested grain will be destroyed by deep burial or with a hammer mill and the milled product ploughed back into the trial sites. The remaining plant material will be disked and ploughed. On completion of the trials, the trial sites will be monitored for volunteer maize and any volunteers destroyed before flowering.

(x) The arrangements for transporting the GMO to the release site.

The MON 87460 x NK603 seed will arrive at OR Tambo International Airport in sealed containers. The packaging will be clearly labelled to enable easy identification of the contents. After clearing customs, the seed will be directly transported via courier service to Monsanto’s research facility near (DELETED: Section 68(a), (b) and (c) ii of the Promotion of Access to Information Act). At (DELETED: Section 68(a), (b) and (c) ii of the Promotion of Access to Information Act) the seed will be unpacked and re-packed into labelled planting envelopes before being transported to the trial sites. Thereafter the seed will be secured and handled only at the trial location. Any seed remaining after planting of the trial will be destroyed or sent for storage at the (DELETED: Section 68(a), (b) and (c) ii of the Promotion of Access to Information Act) or at the (DELETED: Section 68(a), (b) and (c) ii of the Promotion of Access to Information Act). Accurate records of these procedures 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 – this application is for a field trial.

10) Monitoring and accidents (i) Indicate the methods and plans for monitoring of the GMO

Monsanto will assign a dedicated person to actively manage and monitor the trial at each of the locations. In addition to the data pertaining to B. fusca damage, data pertaining to standard agronomic and phenotypic traits such as root lodging, stalk lodging, yield, grain moisture, plant



height, stand count, barrenness and leaf firing will also be captured during the trial. The dedicated person at each trial site would be responsible for communicating any required information to the Registrar and attending to 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. On completion of the trials, the trial sites would be left fallow or rotated to another crop to enable monitoring for volunteer maize. Any volunteers will be 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 circumstances or effect.

(ii) Indicate any emergency procedures that will be applied in the event of an accident Monsanto does not anticipate any hazardous or deleterious effects arising from field trials with MON 87460 x NK603. Considering that no other effects are expected during field trials with this event, Monsanto does not anticipate additional monitoring requirements other than that which would be required during the trials and compliance with permit conditions. However, should any unanticipated effects be detected during the trial these will be noted, monitored and assessed as the trial progress. Data pertaining to standard agronomic traits such as root lodging, stalk lodging, yield, grain moisture, plant height, stand count, barrenness and leaf firing will be captured and phenotypic development, disease resistance, insect presence and activity will be observed during the trials. As maize is most sensitive to drought stress during flowering and grain fill, three water-availability scenarios will be evaluated to compare MON 87460 x NK603 to its non-transgenic control: (1) Optimal water availability (2) Intermediate stress – drought stress affecting grain fill and (3) Severe stress – drought stress affecting flowering and grain fill. 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.

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 these trials will be imported under the required permits and phytosanitary certificates.



Monsanto has been conducting maize field trials in South Africa for more than 15 years, with no ecologically disruptive impacts recorded as a result of these trials. In addition, as there are no wild relatives of maize in SA and maize, including MON 87460 x NK603, requires intensive human intervention to succeed, no ecologically disruptive impacts are anticipated during these field trials.

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 x NK603 is equivalent to conventional maize and would be handled in the same manner as conventional maize. No additional 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 x NK603 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 (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.



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 Bevan, M., W.M. Barnes and M. Chilton. 1983. Structure and transcription of the nopaline synthase gene region of T-DNA. Nucl. Acids Res. 11: 369-385. Brown, S.M. and C.G. Santino. 1995. Enhanced expression in plants. United States Patent No. 5,424,412. Donovan, W.P. 1991. CryIIB crystal protein gene from Bacillus thuringiensis. United States Patent No. 5,073,632. 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 Kay, R., A. Chan, M. Daly and J. McPherson. 1987. Duplication of CaMV 35S promoter sequences created a strong enhancer for plant genes. Science. 236: 1299-1302. Klee, H.J., Muskopf, Y.M. and Gasser, C.S. (1987) Cloning of an Arabidopsis thaliana gene encoding 5-enolpyruvylshikimate- 3-phosphate synthase: sequence analysis and manipulation to obtain glyphosate-tolerant plants. Mol. Gen. Genet., 210, 437-42. Lamppa G., G. Morelli and N. Chua. 1985. Structure and developmental regulation of a wheat gene encoding the major chlorophyll a/b-binding polypeptide. Mol.Cell. Biol. 5: 1370-1378. 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 Matsuoka, M., Y. Kano-Murakami, Y. Tanaka, Y. Ozeki and N. Yamamoto. 1987. Nucleotide sequence of the cDNA encoding the small subunit of ribulose-1,5-bisphosphate carboxylase from maize. J. Biochem. 102(4): 673-676.

http://www.efsa.europa.eu/EFSA/efsa_locale-1178620753812_1211902604575.htm

http://www.efsa.europa.eu/EFSA/efsa_locale-1178620753812_1211902604575.htm



McElroy, D., A.D. Blowers, B. Jenes and R. Wu. 1991. Construction of expression vectors based on the rice actin 1 (Act1) 5’ region for use in monocot transformation. Mol. Gen. Genet. 231: 150-160. McElroy, D., Zhang, W., Cao, J. and Wu, R. (1990) Isolation of an efficient actin promoter for use in rice transformation. Plant Cell, 2, 163-171. McElwain, E. and S. Spiker. 1989. A wheat cDNA clone which is homologous to the 17 kd heat-shock protein gene family of soybean. Nucl. Acids Res. 17: 1764. Odell, J.T., F. Nagy and N. Chua. 1985. Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter. Nature. 313: 810-812. Padgette, S.R., Re, D.B., Barry, G.F., Eichholtz, D.E., Delannay, X., Fuchs, R.L., Kishore, G.M. and Fraley, R.T. (1996) New weed control opportunities: development of soybeans with a Roundup Ready gene. CRC Handbook, 4, 53-84. 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 Rogers, S.G. 2000. Promoter for transgenic plants. United States Patent No. 6,018,100. Sambrook, J. and D. Russell 2001. “Molecular Cloning” third edition, Cold Spring Harbor Laboratory Press. Chapter 5, Protocol 1: Agarose gel electrophoresis. 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 Widner, W.R. and H.R. Whitely. 1989. Two highly related insecticidal crystal proteins of Bacillus thuringiensis subsp. kurstaki possess different host range specificities. J. Bacteriol. 171(2): 965-974 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



PART II COMMON FORMAT FOR RISK ASSESSMENT

(In accordance with Annex III of the Cartagena Protocol on Biosafety)

Risk assessment details

1. Country Taking Decision:

South Africa

2. Title: MON 87460 x NK603 trial release application

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 and hybrids containing MON 87460 x NK603

5. Unique identification of the living modified organism:

For MON 87460 the unique identifier is MON-8746Ø-4

For NK603 the unique identifier is MON-ØØ6Ø3-6

6. Transformation event: MON 87460 x NK603

7. Introduced or Modified Traits:

A. Abiotic environmental tolerance - Drought or water tolerance B. Altered growth, development and product quality Chemical tolerance - Herbicide tolerance

8. Techniques used for modification:

MON 87460 x NK 603 was obtained through traditional breeding of maize inbred lines, one containing MON 87460, the other containing NK 603. The F1 hybrid seed (MON 87460 x NK 603) thereby inherits the abiotic stress tolerance trait from MON 87460 and the glyphosate tolerance trait from NK 603. The technique used to produce MON 87460 x NK 603 was traditional breeding.



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 by Agrobacterium-mediated transformation of embryonic maize cells.

Maize event NK 603 was developed through the introduction of a glyphosate tolerant 5-enolpyruvyl shikimate-3-phosphate synthase (epsps) gene derived from Agrobacterium sp. strain CP4 (cp4 epsps), conferring tolerance to herbicide products containing glyphosate.

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. The plasmid vector PV-ZMGT32L, containing the EPSPS gene is derived from Agrobacterium sp. strain CP4 (CP4 EPSPS).

11. Insert or inserts (Annex III.9(d)):

As indicated above, MON 87460 x NK 603 was obtained through traditional breeding of maize inbred lines, one containing MON 87460 and the other containing NK 603. The stacked product MON 87460 x NK 603 therefore expresses the cspB and CP4 EPSPS proteins, resulting in MON 87460 x NK 603 maize hybrids being protected against abiotic stress and being tolerant to herbicides containing glyphosate.

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 Maize

14. Point of collection or acquisition of recipient or parental organisms:

MON 87460 x NK 603 was produced using elite parent lines into which the MON 87460 and NK 603 events have been introgressed independently, using conventional breeding techniques. The original transformations that produced the individual events used privately owned germplasm acquired for this purpose.



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.



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, suropical 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).

17. Centres of genetic diversity, if known, of recipient organism or parental organisms:

Refer to 16 above



18. Habitats where the recipient organism or parental organisms may persist or proliferate:

As noted by OECD (2003), maize is not invasive of natural habitats, does not persist or disperse anywhere in the world without the 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. The bulk of the 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°C 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)

The donor organism used to develop the single event MON 87460 was Bacillus subtilis

The donor organism used in development of the single event NK 603 was Agrobacterium tumefaciens strain CP4

20. Common name of donor organism(s):

These organisms are ubiquitous in nature

21. Point of collection or acquisition of donor organism(s):

These organisms are ubiquitous in nature

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 objective of the application is for the trial release of maize containing MON 87460 x NK603 in South Africa. If successful confined field trials will be conducted at Orania, Hopetown, Lutzville and Malelane.

24. Receiving environment (Annex III.9(h)):

The receiving environments are varied and represent the maize producing area in South Africa. There are no reported and scientifically documented cases of any impact on biodiversity or animal and human safety concerns.



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. Detection methods for NK603 has been provided in a similar fashion.

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 x NK603 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 17 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 x NK 603 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.

No potentially adverse effects were detected based on extensive characterization of MON 87460 x NK 603, which included molecular analysis, expression analysis, compositional analyses and phenotypic evaluation.

Testing of MON 87460 x NK 603 demonstrated no changes in its ability to persist in the environment without human intervention or to become invasive compared to conventional maize. As such, the potential consequences to biodiversity resulting from MON 87460 x NK 603 for commercial use, including food, feed or processing, are the same as with conventional maize.

Any volunteer seed germinating in subsequent growing seasons would be detected in the fields that were planted and destroyed using chemical or mechanical means.

28. Overall risk (Annex III.8(d)):

Considering the potential risks and the consequences should the potential risks materialize, the overall risk of cultivating/field testing MON 87460 x NK603 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.

30. Actions to address uncertainty regarding the level of risk (Annex III.8(f)):

The potential risks for the specific product is 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 regarding the safety of MON 87460 x NK603 is contained in the application preceding this section. Additional information can be obtained from the individual event dossiers submitted for MON 87460 and NK603.

32. Any other relevant information:

None

33. Attach document: Not applicable to applicant

34. Notes: None

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.



Attachment A

PRELIMINARY ANNUAL TRIAL REPORT

(DELETED: Section 68(a), (b) and (c) ii of the Promotion of Access to Information Act)

ATTACHMENT B

Trial site information

(DELETED: Section 68(a), (b) and (c) ii of the Promotion of Access to Information Act)

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