Biosafety Assessment of Genetically Modified Plants...
Transcript of Biosafety Assessment of Genetically Modified Plants...
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: [email protected]
*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
e
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