Biosafety Assessment of Genetically Modified Plants: Technical methods of the biosafety assessment for

genetically modified sugarcane*

Bambang Sugiharto Laboratory for Molecular Biology and Biotechnology

Center for Advanced Science and Technology, University of Jember Jl. Kalimantan No 37, Tegalboto Campus, Jember 68121 Indonesia

Email: sugiharto.fmipa@unej.ac.id

*Presented at the International Seminar and Workshop on Agricultural Biotechnology and Biosafety, University of Jember, 10-11 July 2019

Introduction

Modern agricultural biotechnology provides a new technique in the development of a new plant cultivar

Genetic engineering may modulate biochemical pathway which may lead to undesirable effect on human health and environment

The genetically modified (GM) plants must follow regulation determined by national authority before entering into market.

The Indonesian government regulation (PP) Number 21, 2005 is the regulation for Biosafety of Genetic Engineering Product

Biosafety assessment (a) environments; (b) food; (c) feed – safety assessments.

Genetic Modified Plant is another breeding method

Poor tomato but disease resistant

Elite, disease resistant tomato

Recombinant DNA (or GM) allows a single gene to be

introduced into a genome. This method can be faster than

conventional breeding

Elite tomato

Genetically Modified (GM) Tomato

Outline Biosafety Assessment : Case study on biosafety assessment of GM sugarcane

Environmental safety

Food safety

Feed safety

Document Evaluation by Technical Teams

Posting in Biosafety Clearing House

Certification

All of the documents should be proved by scientific evidence

The Environmental Risk Assessment (ERA) The GM sugarcane was grown in confined field trial (LUT)

The GM sugarcane was cultivated with different planting period to protect any possibility to cross hybridization within the family.

Sugarcane cultivars has poor flowering and pollen quickly died

Sugarcane seed is very small and no possible to germinate in cultivated field naturally.

Layout map of confined field trial

The Environmental Risk Assessment (ERA) Interaction with non-target organism - insect biodiversity

Sticky Trap

Pan Yellow Trap

Pitfall Trap

3 Months

GM and Non-GM Sugarcane

Diversity Indeks Shannon-Weinner

Evenness Indeks

6 Months

9 Months

Coleoptera Diptera Diptera Orthoptera

Hymenoptera Orthoptera Hymenoptera Araneae

Total, N-fixing and P-solublizing bacterias

Gene Flow

GM Sugarcane cultivated in

confined field trial

Electroforesis Analysis of DNA PCR Product

Soil sample

Dissolved in PBS

Inoculation on specific media

ERA: Microorganism diversity and the possibility gene transfer from plant to microorganism

0 -

30

cm

R

hiz

osp

her

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Colony PCR GM field Control GM field

Soil fertility

Carbon and mineral content: C, N, P, K, Ca, Mg, S

ERA: Invasiveness Competitive Ability of GM sugarcane toward dominat weed

Replacement Series Method : The competition ability of GM sugarcane toward

competitors (main weeds in sugarcane) Crowding coefficient of GM sugarcane GM sugarcane did no have the potency of invasiveness

Description: Each treatment was repeated three times, making 55 treatments x 3 replicates = 165 experimental pots Description: D: GM sugarcane NT: non-GMO sugar cane X: nut-grass Y: purslane

Food Safety Assesment : Allergenicity and toxicity Aassessment of GM sugarcane

To provide safety for uses as food and/or

feed of GM sugarcane

1. Allergenicity Assessment

2. Toxicity Assessment

Acute Toxicity

LD50, Index Organ

3. Proximate Analysis

• Ash • Crude Protein • Crude Lipid • Crude Fiber, etc

Framework of allergenicity assessment in GM sugarcane

Sequence of Amino Acid

80-Mers Aligment using

Alergenonline.org

Bioinfomatics In Vitro: Thermal and Digestion Stability

Treat protein using SGF (Pepsin) and

SIF (Trypsin)

Detection using SDS PAGE and

Enzimatic Activity

Treat animal using sugarcane juice

Collect sera of blood rat

Detection using Kit ELISA IgE

In Vivo Allergenicity Assay

Day 7

Day 14

Heat treatment 28-60-90ºC

Detection using SDS PAGE & Enzimatic

Activity

Day 0

Day 7 Day 14

Bioinformatics analysis Amino Acid Sequence of SPS

• Bioinformatics analysis was carried out to determine potential allergen and cross-reactivity of SPS and any known allergen proteins, can be identified via similarity to any known allergens.

• The amino acid sequence of SPS compare with those of allergenic proteins using AllergenOnline (databasehttp://www.allergenonline.com, version 18B) • The results shown that no more than 35% shared identity over ≥80 amino acids segments.

• Thus, sugarcane SPS is not potentially allergenic since there was no similarity with allergenic proteins in the database

Proved by laboratory analysis

Thermal and digestion stability

Thermal Stability Assessment

• Proteins with high stability in elevated temperature are generally associated with allergenic potential. To Identification of potential protein hazards can be conducted by examining physical properties through a heating stability assessment.

• Western blot analysis shown the SPS protein was still detected at the highest temperature (90 ◦C) of incubation (Figure A).

• The loss of enzymatic activity suggested that SPSs might be denatured and lose their function (Figure B).

Digestion Stability Assessment

The protein samples treated by the method of SGF (Simulated gastric fluid, pepsin) and SIF (simulated intestinal fluid, trypsin)

The total protein was degraded by SGF and SIF assays - no allergenic potential

In vivo allergenicity assay in rats

• Statistical analysis showed that there is no significant difference (p >0.05) between the C−, Non-GM (WT), and GM (SP) at day 7 and 14 after treatments

• But, significantly higher (p< 0.05) in rats fed OVA (C+) and those fed transgenic sugarcane juice at day 14.

• No allergen of GM-sugarcane

• Collect the juice from GM and non-GM (WT) sugarcane. • Animal were divided into 4 groups treatment, 1. Feeding transgenic sugarcane juice doses at 8 g/kg of body weight per day 2. Feeding wildtype sugarcane juice doses at 8 g/kg of body weight per day 3. Positive control rats were sensitized with Ovalbumin weekly for 14 days, 4. Negative control rats were not treated with either sugarcane juice or OVA. • Blood samples were taken by retro-orbital plexus on days 0, 7, and 14, and

then detection of IgE using a rat IgE ELISA kit

Toxicity assessment of GM sugarcane in mice

Day 1-14, Observe sign of toxicity and body weight of mice

Day 14 Index Organ

Dosage Injection • 6.25 gr/kg BB • 12.5 gr/kg BB • 25 gr/kg BB • Control

(CMC/Na)

Sugarcane Juice Transgenic

4 groups treatment (n= 5 male/5 female)

Result of Toxicity Assessment

Acute Toxicity in Mice

Relatively Harmless

Toxicity assessment: body weight and indeks organ of mice

Indeks Organ of Mice

• Statistical analysis showed that there was no significant difference (p > 0.05) in regard to the gain in body weight between sugarcane juice treatments and the control

• The wet weights of the organs showed no significant differences (p > 0.05) between the sugarcane juice and control treatments. The relative organs wet weight of heart, liver, spleen, and kidneys were also similar and not affected by sugarcane juice treatments

• The weight of the mice was also recorded daily for 14 days of observation. • On day 14, the mice were sacrificed via cervical dislocation and dissected to examine

the morphological condition of vital organs including the liver, spleen, kidneys, and heart.

Proposed framework of food safety risk assessment in GM sugarcane

Genetically Modified Sugarcane

Proximate analysis

Leaves Stem

• Moisture • Crude protein • Carbohydrate • Fat • Ash • Crude fibre • Calorie

Juice

Mineral analysis

Leaves Stem

• N • P • K • Ca • Mg

Substantial Equivalent of GM Sugarcane

Substantial equivalent is used to indicate whether the GM plants have similar health and nutritional characteristics with its conventional counterpart.

GM sugarcane has similar nutritional characters, except the targeted trait

Moisture Moisture analyzer

Crude protein

Destruksi Destilator Tritrator Flask Kjeldahl

Crude fat

Flask fat

Ash

Crucible Furnance

Carbohydrtae

Total carbohydrate by

difference

Crude fibre

Enlenmeyer Incubator

Filter paper analytic

Soxhlet extraction

% Carbohydrate: 100-(% protein + % fat + % ash)

An

alys

is M

eth

od

s fo

r Su

bst

anti

al E

qu

ival

ence

Leave and stem sugar cane

Mineral

Weighed

Furnace at 550°C ; 8 h

Disolved use HCl 6 N

Filter

K, Ca, Mg, P

AAS

N

Kjeldahl

P

Spectrofotometer

Leaves Stems

analytic

Furnance

Filter paper

Phosporus

AAS

Nitrogen

Destilator Tritrator

Spectrofotometer

Measurement of Mineral Contents

In vitro digestibility for feed biosafety assessment

Sugar cane leaves

Cut into small pieces

Dried 105°C; 24 h

Rumen fluid & Buffer solution at pH 6,8 pre-heated 39°C

Added CO2

Incubated at 38°C ; 48 h dark condition

Centrifugated 2500 rpm ; 15 minute

Addes pepsin HCl solution 0,2%

Incubated at 38°C ; 48 h dark condition

Centrifugated 2500 rpm ; 15 minute

Filtered with whatman paper

Dried 105°C ; 24 h

Weight

Ashed 450°C ; 6 h

Weight

Added CO2

Leaves Buffer soulution Rumen fluid

Incubate at 38°C;48h

Centrifugate

Fermentor tube

Add pepsin HCL 0,2%

Incubate at 38°C;48h

Centrifugate

Paper filter

Dried 105°C ; 24 h

Ashed 450°C ; 6 h

Weight

CONCLUSION : Biotechnology is needed to improve plant

production which may lead to effect on health and environment

Biosafety assessment should be conducted with scientific evidence to prove that GM plants are safe

Biosafety assessments consist of environmental risk (ERA), food safety and feed safety assessment

ERA is directed to identify invasiveness of GM, no gene transfer, no effect on microorganism and human health.

Food and feed safety assessment mainly are addressed to prove that no allergenicity and toxicity of GM plants

THANKS YOU VERY MUCH TERIMAKASIH

The picture was taken after cultivation of GM sugarcane in Confined Field Trial (LUT), 2016