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SECTION G: PROPOSED EXPERIMENTAL PROGRAM

OVERVIEW:

The proposed experimental use program (EUP) will be under the management of Michael 

Irey, Director of Research and Business Development, Southern Gardens Citrus

Nursery, LLC.

Michael Irey

Director of Research and Business Development

Southern Gardens Citrus Nursery, LLC

1820 County Road 833

Clewiston, FL 33440

863-902-2249

863-902-2272 fax

mirey@southerngardens.com

PURPOSE:

Gather and evaluate samples collected in the field which will be used in analytical studies 

to produce data, under testing outlined in the EUP for the purpose of supporting the FIFRA 

Section 3 registration application for genetically engineered Citrus tristeza virus expressing 

spinach defensin proteins 2, 7, and 8 (GE CTV-SoD).

Constructs containing one, two, or three defensin genes (2, 7 and 8) from spinach will be 

used in this EUP. Additional information on constructs is provided with this submission in 

report ID SGC-039.

There will be multiple EUP locations in Florida citrus producing counties. The total acreage 

proposed for this EUP is 400. Testing will be conducted to generate agronomic, efficacy 

and regulatory data and information:

Total Proposed Acreage: 400 

Planting and design of trials has not been finalized at the time of EUP submission. Actual 

and specific planting information listed under the “Planting/Growing/Harvesting Schedule” 

section will be provided as required in the EPA permit approval letter. As described in the 

accompanying Product Characterization document, SGC will be reporting to EPA the 

exact location of the SoD gene inserts (genetic maps) when final decisions are made 

regarding their location(s) in the vector constructs. 

Calculations are provided on the following page to give a “worst case”, “not to exceed” 

amount of active ingredient that could be expressed in this EUP.

This EUP will be conducted in Florida citrus producing counties only. 

The total amount of SoD2, SoD7 and SoD8 protein expressed in GE CTV that has been 

inoculated into citrus trees for ALL FIELD TESTING in Florida citrus producing counties

included in the EUP WILL NOT EXCEED the following levels based on “worst case” 

assumptions described on the next page: 20 kilograms. This amount is based on the 

following information and following calculation:
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Kgs. of citrus leaf tissue contained per acre of citrus trees

A projected maximum of 20 kg. citrus leaf tissue (dry wt.) per tree is used for this 

calculation. Amounts of dry wt. leaf tissue per citrus tree vary with citrus type. Sweet 

orange has been measured at an average weight of 12 kg dry. wt. leaf tissue for a mixture 

of trees from 3-15 years old (Morgan, 2006)1. We have decided to use 20 kg. as a high 

estimate, which takes into account the fact that grapefruit trees are larger than sweet 

orange trees. 

Maximum number of citrus trees per acre of citrus EUP grove

Citrus tree density: 250 trees/acre2

Maximum amount of GE CTV expressing defensins 2, 7 and 8 protein produced per 

gram citrus tissue

Although no defensin protein has been detected (Limit of Detection = 2.8 ppm dry wt.) in a 

limited number of citrus samples analyzed, to be conservative, SGC will use 10 μg per g 

(dry wt.) citrus tissue in this calculation for “will not exceed” or “worst case” amount of 

defensin protein produced under this EUP. (Reference report ID SGC-039 submitted with 

this application for additional information.)

Calculated estimate of the maximum amount of GE CTV expressing SoD2, SoD7 and

SoD8 proteins produced per acre of inoculated citrus trees:

20,000 g plant tissue/tree • 250 trees/acre • 10 x 10-6 g defensin /g plant tissue • 400 acres 

= 20 kg SoD2 and/or SoD7 and/or SoD8

 1 Morgan et. al. 2006. Size, Biomass and Nitrogen Relationships with Sweet Orange Tree Growth. J.Amer.Soc.Hort.Science 

131(1):149-156. (average amount of leaf tissue (dry wt.) per 6 y.o. tree = 13 kg.) 2

M. Irey. 2014. Personal communication on maximum tree density of 250 trees/acre under EUP in Hendry County, Florida.
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Planting/Growing/Harvesting Schedule:

All field trials conducted under this EUP will be planted between the dates of September 1, 

2016 and August 31, 2019. 

The following planting information will be recorded by Southern Gardens and maintained at 

Southern Gardens Citrus Nursery, LLC, facilities in Clewiston, FL, 33440.

· Event ID 

· Construct map(s) identifying the location of the inserted defensin gene(s)

· Gene(s)

· Citrus type and variety

· Planting location (county, state, Grove and GPS coordinates) 

· Acreage 

· Planting Date 

· Responsible person contact information 

· Cooperator contact information 

This information will be quality assured and provided to the Agency as required in the EPA 

permit approval letter. A spreadsheet (draft attached) will serve as the reporting format.

Conditions of the trials, including isolation and confinement procedures, will be consistent 

with those described in USDA-APHIS-BRS permits (13-039-101r-a2 and others to be 

issued in the future). 

Isolation and Confinement Procedures:

Plots/plantings will be arranged so that there will be two rows of non-CTV-treated trees (or 

the equivalent of two rows of plants in distance down the row) surrounding the regulated 

material (i.e., trees with the GE CTV-SoD). In addition, the distance from the regulated GE 

CTV-SoD trees to the nearest commercial trees will be no less than 200 ft.

In addition, the EUP plots will be posted. Access will be restricted to authorized and trained 

personnel.

The identity and location of disease resistant citrus tree material will be maintained via 

greenhouse and field maps. 

No fruit from any tree under this EUP (trees containing the GE CTV-SoD and the 

surrounding two rows) will be allowed to enter food or feed channels to be used as food or 

feed products without establishment of an exemption from the requirement of a tolerance 

on citrus fruits for GE CTV-SoD. 

Harvest Procedures:

Samples of flowers, leaves and fruit will be taken for analysis. Fruit not analyzed will be 

pulled from the trees and left on the ground to decompose. This fruit will not be allowed to 

enter the food or feed chain without establishment of an exemption from the requirement of 

a tolerance (on a temporary basis).

Final Disposition of Tree Material Covered under the EUP:
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At the conclusion of this EUP, trees included herein will either be destroyed (devitalized), 

or the GE CTV-SoD construct(s) will be registered under FIFRA with appropriate 

tolerances or tolerance exemptions established, allowing the trees to remain in the fields. 

Devitalization will consist of cutting down trees, allowing time for desiccation, followed by 

burning of the trees. Stumps will be treated with an appropriate herbicide.

Post-Harvest/Destruction Monitoring Procedures:

In sites where trees have been cut down, the stumps will be monitored on a monthly basis 

for sprouting. If sprouts are detected, they will be cut and the stump with the sprouts will 

be treated again with herbicide. The site will be monitored for 1 year after the trees are 

removed.
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Proposed Testing to Generate Agronomic, Efficacy and Regulatory Data and 

Information 

(Proposed 400 acres)

Objectives:

The objectives of all field testing under this Experimental Use Permit are to: 

1. Generate data which will be used to support a FIFRA Section 3 registration and 

permanent exemption from the requirement of a tolerance for genetically engineered 

Citrus tristeza virus expressing spinach defensin proteins 2, 7 and 8 (GE CTV-SoD). 

These experiments will include the following:

a. Evaluate levels of resistance and/or tolerance to CG for each construct tested.

b. Measure expression of GE CTV-SoD in plant tissues (e.g., leaf, flower, pollen, immature 

fruit, mature fruit). For this experiment, leaves, flowers and pollen, as well as fruit will be 

sent to an appropriate laboratory for quantitative analysis.

c. Evaluate potential effects of disease resistant citrus on non-target organisms. Visual 

observations of NTOs -- including honeybees -- will be recorded. 

d. Evaluate compositional and nutritional data in raw and processed citrus commodities 

produced from GECTV-SoD. Samples may be sent to an appropriate laboratory for 

determination of proximate as well as nutrient levels.

2. Generate Data for to Support a FIFRA Section 3(c)(7)(C) Public Interest Finding: 

a. Agronomic data to demonstrate that trees inoculated with GE CTV-SoD produce citrus 

fruits of comparable size and volume, and at comparable rates of growth, to those of 

standard conventional commercial trees. Data points include, but are not limited to, the 

following: tree growth rates, vigor, maturation, flower and fruit production (as feasible).

b. Evaluate the general susceptibility of citrus trees inoculated with GE CTV-SoD to disease 

pressure as compared to conventional commercial citrus trees.

c. Comparative data to demonstrate that fruit from citrus trees inoculated with GE CTV-SoD 

is of comparable quality to conventional fruit. Refer to 1.d

No adverse effects on any trees, non-target organisms, or the environment are anticipated 

under this EUP. Information received regarding any adverse finding under this EUP will be 

considered and reviewed for reportability to the Agency. 
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Responsible Participants:

Names and contact information of participants will be recorded and maintained by Southern 

Gardens, and will be provided to the Agency as required in the EPA permit approval letter. A 

spreadsheet will serve as the reporting format.

Trials for Data Collection:

Young and established citrus trees will be inoculated with GE CTV expressing one, two, or 

three spinach defensin proteins 2,7 and 8. Experimental design will be focused on gathering 

data on the level of disease resistance to infected trees using two designs:

1) Citrus tree seedlings, sweet orange, grapefruit, navel orange and/or lemons will be 

inoculated via grafting in the greenhouse with GE CTV expressing one, two, or three

spinach defensin proteins. Once infection with GE CTV is confirmed (PCR or ELISA), 

the trees will be planted to the field. It is anticipated that the trees will become infected 

with CG (HLB) and citrus canker via natural vector transmission.

2) Healthy and naturally CG infected trees in the field will be inoculated with GE CTV 

SoD by graft inoculation and monitored for time to infection and recovery from CG 

symptoms.

In both designs above, no inoculation for citrus canker will occur, but trees will be exposed 

naturally to this disease. In experiment 1) resistance to natural infection from CG (HLB) will be 

monitored and observed. In experiment 2), recovery from the disease is the goal.

Spinach defensin proteins have been shown to be effective in the control of citrus greening 

(CG) also known as Huanglongbing (HLB). GE CTV-SoD is inoculated into citrus trees already 

diseased with HLB, in an effort to restore tree health; or, is intended to prevent losses caused 

by HLB by inoculating healthy trees with the modified CTV vector prior to putting them in the 

field. The purpose of this EUP is to gather additional data from multiple constructs of GE CTVSoD.

Neighboring or adjacent conventional citrus trees will serve as comparators. The experiments 

will be established in one of the following: a completely randomized design, a randomized 

complete block, or, in a split-plot design. Plot size will vary and replications will be conducted. 

Data will be collected over a multi-year period (to be determined based on date of first 

flowering). Data will be analyzed using methods appropriate to the parameter being 

evaluated. Statistical analyses will be performed by analysis of variance (ANOVA) 

methodology using SAS.

Statistical analyses will be run using an appropriate method (e.g. Student’s t-test). 
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Data to be collected for the trials will include:

1) Phenotypic data

a. Appearance

b. Growth measurements

2) Production data 

a. Date to first flowering 

b. Expression levels of spinach defensin proteins in citrus tissues and 

commodities

c. Fruit yield (as feasible)

d. Fruit and juice quality 

3) Resistance data

a. % citrus greening infection

b. Symptom severity

4) Other data

a. Insect tolerance/attractiveness

b. Other disease resistance data (e.g. citrus canker)

c. Any other observations as appropriate.