Greening-Resistant Genetically-Enhanced Trees: A case study

Michael S. Irey

Huanglongbing – aka Citrus Greening

• Found in Florida in

2005

• Now found along with

it’s vector in:

– Florida (endemic)

– Georgia

– South Carolina

– Louisiana

– Texas

– California

• Considered to be the

worst disease of citrus

• Bottom line

– Production costs up

40-50%

– Production losses

– Possible effects on

quality

– Current management

efforts are not

sustainable……..

Varietal Resistance

Most Susceptible

Most Tolerant

•Poncirus trifoliata,

citranges

•Grapefruit, lemons,

pummelos, sour orange

•Sweet oranges,

mandarins, tangelos

Varietal Resistance

Bottom line: No real resistance

in Citrus sp.

Sustainable Management

• Current pesticide usage can’t continue

• Florida nursery capacity can’t support increased

tree mortality or increased roguing as trees begin

to decline

• Varietal resistance

– Must be part of a long term strategy

– Resistance is generally thought to be foundation of a

sustainable management program

Varietal Resistance

• How do we get there?

– Traditional breeding

• Need to identify adequate sources of resistance and then

transfer the traits to commercial types

• Very long term approach (20+ years)

– Biotechnology

• Short to medium term approach

• The science may be the easy part…..

• Case study – Development of a resistant plant

using a gene from Spinach

Major steps

• Efficacy

– Which gene?

– How do you test?

• Greenhouse

• Field

– Regulations

• Federal

• State

• Freedom to operate

– Genetic elements

– Methods

• Regulatory

– USDA

– EPA

– FDA

• Cost of the projects

and timeline

Efficacy

• Which gene?

– Want one that is not

objectionable

– Not toxic to nontarget

organisms

– Not allergenic

– No consumer

acceptance issues

• How do we test for

resistance?

• SGC Project – Gene

from Spinach

– Already in the food

chain

• Contrasted with genes

from human or pig

– Not allergenic

• Contrasted with genes

from crustacean and bee

venom

– Generally well received

because it is spinach

– Toxicity?

Efficacy

• How do we test?

– Field trial vs. greenhouse trials

– If field trial, how long will it take

– If greenhouse trial, how will we inoculate and will it be

representative of field resistance

– How do we get agronomic performance and yield data

• Juvenility

• Can’t flower

• Trees/technology originates in Texas

– Florida quarantine procedures

– Had to develop a process to import to Florida

Efficacy

• Field trial

– USDA permit

– Yearly reporting

requirements

– Yearly inspections

– Land intensive

– Expensive

– Carries $$$ penalties

for violations

– Time consuming

• 3 years to get data

1 Mile radius = ~2,010 ac

Efficacy

• Field trial

– USDA permit

– Yearly reporting

requirements

– Yearly inspections

– Land intensive

– Expensive

– Carries $$$ penalties

for violations

– Time consuming

• Many years of data

collection

Efficacy

• Field trial

– USDA permit

– Yearly reporting

requirements

– Yearly inspections

– Land intensive

– Expensive

– Carries $$$ penalties

for violations

– Time consuming

• Many years of data

collection

Special considerations…….

A serious incident, or a history of lesser incidents, may prompt BRS to refer the

situation to APHIS’ Investigative and Enforcement Services (IES) for further

investigation. BRS also works closely with State Departments of Agriculture and

other federal agencies, including the Food and Drug Administration, the

Environmental Protection Agency and the Department of Justice, to ensure

compliance with APHIS regulations. The Plant Protection Act provides for

substantial penalties for serious infractions, including fines up to $500,000 and the

possibility of criminal prosecution.

Efficacy

• Greenhouse testing

– Can test many more

lines

– Quicker (less than a

year)

– Little permitting

required

– Less inspections

– Needs a dedicated

facility: very expensive

Efficacy

• “Death house”

– Material must be tested

for all diseases of

concern before arrival

– Must be inspected on

arrival

– Once in the “death

house”, can never be

released

– No propagations other

than for testing

Efficacy

Freedom to operate

• Multiple things to consider

– Process used to make transgenics

– Actual genetic elements

– Tweaks

– Process involved in conferring resistance

Selectable element Gene of interest

Promoter (switch to turn it on)

Gene (antibiotic selection, functional gene)

Terminator (switch to turn it off)

Leader (enhancing sequence)

* * *

Freedom to operate

• Agrobacterium

transformation

– Syngenta

• Codon optimization

– Better expression

– Various

Regulatory

• PIP – Plant incorporated protectant

“Plant-incorporated protectants are pesticidal

substances produced by plants and the genetic material

necessary for the plant to produce the substance”

• Will be regulated by:

– EPA

– USDA

– FDA (voluntary)

Regulatory – Roles of the agencies

• EPA regulates the sale, distribution, and use of

pesticides in order to protect health and the

environment (EUPs)

• USDA (APHIS) is responsible for protecting

agriculture from pests and diseases and has

regulatory oversight over products of modern

biotechnology that could pose such a risk

• FDA is responsible for ensuring the safety and

proper labeling of all plant-derived foods and

feeds, including those developed through

biotechnology

Regulatory - EPA

• Need to establish the safety of the PIP

– Tolerance or tolerance exemption

• Currently done by “event”

– Trying to work towards by “construct”

– Trying also to work toward inclusion of all citrus

• Why is this important? • Scions (Hamlin, Valencia, Navel, Lemon, Red Grapefruit,

White Grapefruit, Murcott, etc.)

• Rootstocks (Carrizo, Swingle, C35, Sour Orange, etc.)

• Data requirements

Regulatory - EPA

• Required data for a

tolerance

exemption/EUP

– Some we will

petition to be

exempt from

– Some in progress

– All must be

addressed

– All are expensive

(ex. $250K for 0.5g)

Guideline Ref. Description

151-20 Product Identity

151-20 Manufacturing Process

151-22 Discussion of Formulation of Impurities

151-23 Analysis of Samples

151-25 Certification of Limits

151-27 Submittal of Samples

151-30 Acute oral Toxicity

151-31 Subchronic Oral Toxicity

154-16 Avian Acute Oral Toxicity

154-17 Avian Dietary Toxicity

154-18 Wild Mammal Toxicity

154-19 Fish Toxicity

154-20 Freshwater Aquatic Organism Toxicity

154-21 Estuarine and Marine Organism Toxicity

154-22 Non-target Plant Studies

154-23 Non-target Insect Studies

154-24 Honeybee Acute Toxicity

Non-Guideline Product Characterization: Plasmid Map & Transformation

Non-Guideline Product Characterization: Insert Integrity

Non-Guideline Product Characterization: Copy Number

Non-Guideline Equivalency of SoD2 + SoD7 proteins (GE citrus v native)

Non-Guideline Product Characterization: Quantitative ELISA Analysis in Plant Tissues

Non-Guideline ELISA Method Validation

Non-Guideline Product Characterization: Inheritance Patterns

Non-Guideline Product Characterization: Event Specific PCR

Non-Guideline Product Characterization: Protein Efficacy and Activity

Non-Guideline In-vitro Digestion/ Thermolability

Non-Guideline Potential for Allergenicity – Bioinformatics

Non-Guideline Equivalency of SoD2 + SoD7 proteins (GE citrus vs. recombinant)

Regulatory - EPA

• In theory, the EPA does not have a problem with

the “by construct” approach, however…..

– We would have to prove that no new ORFs were

created (i.e. no new proteins were produced by

mistake)

– If there were, then we would have to go through the

process with the new proteins as well or START

OVER

Regulatory - USDA-APHIS

• In addition to providing permission and

regulating the field trials, APHIS is the agency

that would issue the document that evaluates

whether the proposed Federal action would

have significant effects on the environment.

• EA, EIS, and FONSI

– EA Environmental Assessment

– EIS Environmental Impact Statement

– FONSI Finding of No Significant Impact

Regulatory – USDA-APHIS

• An EA is a concise document that briefly provides

evidence and analysis for determining the environmental

impacts of a proposed Federal action and alternatives.

• An EIS is a detailed written statement on the

environmental impact of a proposed action; it is prepared if

significant impacts are likely and if an agency is proposing

a major action that significantly affects the quality of the

environment.

• A FONSI is a document that presents the reasons why the

agency has concluded that there are no significant

environmental impacts projected to occur upon

implementation of a proposed action.

Regulatory – USDA-APHIS

• 2004 Petition from

Monsanto for

Nonregulated status

• APHIS conducted an

EA January, 2005

• 9 Months later sued by

organic growers

• Dec 2009 APHIS

published draft EIS

• Final EIS Dec 2010

Regulatory – USDA-APHIS

We are assuming that an

EIS will be needed

89

265

10

34

965 77

196

191

Regulatory – USDA-APHIS

• Three choices in

response for a petition

for non regulated

status:

– Take no action

– Determine that the

article is no longer

regulated on the whole

– Determine that the

article is no longer

regulated in part

• Based on the

above information,

APHIS has considered that C5

HoneySweet plum is not a plant

pest and herby approves APHIS

pettition no. 04-264-01p.

Therefore, C5 HoneySweet

plums will no longer be

considered regulated articles

under the regulations at 7 CFR

part 340.

Regulatory - FDA

• Voluntary – In the 1992 policy, FDA

recommended that

developers consult with

FDA about bioengineered

foods under development;

since issuance of the 1992

policy, developers have

routinely done so.

– So in effect, nothing goes

commercial without going

through the FDA process

– These procedures describe

a process in which a

developer who intends to

commercialize a

bioengineered food meets

with the agency to identify

and discuss relevant safety,

nutritional, or other

regulatory issues regarding

the bioengineered food and

then submits to FDA a

summary of its scientific

and regulatory assessment

of the food; FDA evaluates

the submission and

responds to the developer

by letter

Regulatory - FDA

• The name of the bioengineered food and the crop from which it is

derived.

• A description of the various applications or uses of the bioengineered

food, including animal feed uses.

• Information concerning the sources, identities, and functions of

introduced genetic material.

• Information on the purpose or intended technical effect of the

modification, and its expected effect on the composition or

characteristic properties of the food or feed.

• Information concerning the identity and function of expression products

encoded by the introduced genetic material, including an estimate of

the concentration of any expression product in the bioengineered crop

or food derived thereof.

• Information regarding any known or suspected allergenicity and

toxicity of expression products and the basis for concluding that foods

containing the expression products can be safely consumed.

• Information comparing the composition or characteristics of the

bioengineered food to that of food derived from the parental variety or

other commonly consumed varieties with special emphasis on

important nutrients, and toxicants that occur naturally in the food.

• A discussion of the available information that addresses whether the

potential for the bioengineered food to induce an allergic response

has been altered by the genetic modification.

• Any other information relevant to the safety and nutritional

assessment of the bioengineered food.

Regulatory - FDA

Regulatory - FDA

• Series of

“consultations”

– Unofficial

– Official

• Initial Consultations

– Resolution of safety,

regulatory and

nutritional issues

– Multiple

• Final Consultations

– Submit and defend

• What you are looking

for is a letter of “Not

materially different in

composition, safety,

wholesomeness, or

any relevant

parameter….”

Regulatory - FDA

• Intended effect

• Regulatory considerations

• Genetic modifications

• Food/Feed use

• Compositional analysis

Regulatory - FDA

• Intended effect

• Regulatory considerations

• Genetic modifications

• Food/Feed use

• Compositional analysis

USDA-ARS has concluded that plum line

C5 is not materially different in

composition, safety, wholesomeness, or

any relevant parameter from plums now

grown, marketed, and consumed.

At this time, based on USDA-ARS's data

and information, the agency considers

USDA-ARS's consultation on plum line C5

to be complete.

Regulatory - FDA

• In theory, FDA does not have a problem with the

“by construct” approach, however…..

– With each new plant, you would have to prove that the

new plant is “substantially equivalent” to the one that

received the letter of “not materially different”

– So, some of the testing is eliminated but there would

still be a significant amount of data that would be

needed

Consumer Acceptance

• After the EPA, FDA and USDA, we now know it is

safe for the environment, people, and animals,

but…..

– We have to get over some other things:

• Source of the gene is important:

– Crab

– Human

– Pig

• How it is presented is important

– Nice to have vs. necessary

– Other benefits

Consumer Acceptance

• Arctic Apple

– Uses gene sequences actually derived from apple

– Resistant to enzymatic browning (don’t brown when

bruised, sliced, etc.)

1932 Submissions

Most were negative

Consumer Acceptance

Consumer Acceptance

•It has to be done right……

Recovery of the Investment

• Citrus is a vegetatively propagated crop

• Nursery tree approach: charge a technology access

fee at the point of sale

– ~3,000,000 trees available to growers per year

– Probably can’t charge too much for trees or it may be cost

prohibitive

– One time shot

• Based on production

– No system to capture value

– If good for grower, then good for tree supplier

– More potential for value recovery

• Hybrid

TBD

So with all of this being said….

• We are moving forward……

– Think we have an acceptable gene (consumer)

– Initial data shows that some lines are resistant

– We are producing synthetic peptide and working on

equivalency

– Starting bee toxicity testing

– Starting to generate data for EUP and tolerance

exemption

– Have done the freedom to operate due diligence and

have acquired rights to necessary technology

It is a daunting task, but the end goal is in sight….