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
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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
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
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
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