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Modified Organism
IND-ØØ41Ø-5 x MON-Ø4Ø32-6 - Drought and Glyphosate tolerant soybean
Record information and status
Record ID
115071
Status
Published
Date of creation
2019-07-29 15:08 UTC (austein.mcloughlin@cbd.int)
Date of publication
2019-07-29 15:08 UTC (austein.mcloughlin@cbd.int)

Living Modified Organism identity
The image below identifies the LMO through its unique identifier, trade name and a link to this page of the BCH. Click on it to download a larger image on your computer. For help on how to use it go to the LMO quick-links page.

LMO name
Drought and Glyphosate tolerant soybean
Transformation event
HB4 x GTS 40-3-2 (40-3-2)
Unique identifier
IND-ØØ41Ø-5 x MON-Ø4Ø32-6
Developer(s)
Bioceres S.A.
Ocampo 210 bis
Predio CCT Rosario
(2000) Rosario, Santa Fe, Argentina
Tel: (54) 341 4861100
Rosario
Argentina
Phone:+ 54 341 4861100
Url:Bioceres (INDEAR)
Verdeca
Ocampo 210 bis, predio CCT
Rosario, Santa Fe
Argentina, 2000
Phone:(54) 341 486 1100
Email:geronimo.watson@indear.com
Url:Verdeca
Description
Soybean (Glycine max) was produced through crossing Verdeca HB4 Soybean and Roundup Ready™ soybean. The stacked modifications confer tolerance to drought conditions and glyphosate herbicides through the expression of sunflower (Helianthus annuus) homeobox-leucine zipper protein 4 (HaHB4) and Agrobacterium tumefaciens 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) proteins, respectively.

Note: The Streptomyces hygroscopicus bar gene was also inserted into the genome and was used for selection during transformation (not with the intention for herbicide resistance).
Recipient Organism or Parental Organisms
The term Recipient organism refers to an organism (either already modified or non-modified) that was subjected to genetic modification, whereas Parental organisms refers to those that were involved in cross breeding or cell fusion.
MON-Ø4Ø32-6 - Roundup Ready™ soybean
Resistance to herbicides - Glyphosate
Show detection method(s)
IND-ØØ41Ø-5 - Verdeca HB4 Soybean
Resistance to herbicides - Glufosinate Tolerance to abiotic stress - Drought, Salinity
Show detection method(s)
Point of collection or acquisition of the recipient organism
IND-ØØ41Ø-5 - Glycine max cv. Williams 82

MON-Ø4Ø32-6 - Line: A5403
Characteristics of the transformation process
Vector
pIND2-HB4 & PV-GMGT04
Techniques used for the modification
  • Cross breeding
Genetic elements construct
 
Vegetative storage protein terminator
0.55 Kb
 
 
Phosphinothricin N-acetyltransferase gene
0.56 Kb
 
 
TEV 5' Untranslated Region
0.13 Kb
 
 
CaMV 35S promoter
0.34 Kb
 
 
CaMV 35S promoter
0.34 Kb
 
 
Homeodomain-leucine zipper 4 promoter
1.21 Kb
 
 
Homeodomain-leucine zipper 4 gene
0.53 Kb
 
 
Nopaline Synthase Gene Terminator
0.25 Kb
 
 
CaMV Enhanced 35S promoter
0.61 Kb
 
 
Chloroplast Transit Peptide 4
0.23 Kb
 
 
5-enolpyruvylshikimate-3-phosphate synthase gene
1.36 Kb
 
 
Nopaline Synthase Gene Terminator
0.26 Kb
 
Further details
Notes regarding the genetic elements introduced or modified in this LMO
Genetic elements introduced from pIND2-HB4
bar gene cassette (Anti-sense orientation)
The transcription of the Streptomyces hygroscopicus phosphinothricin N-acetyltransferase (bar; PAT protein) gene is under the control of the duplicated Cauliflower Mosaic Virus (CaMV) 35S promoter and the Glycine max vegetative storage protein terminator. To enhance translation, the Tobacco Etch Virus 5' untranslated region was included at the 5' end of the bar transcript.

Homeodomain-leucine zipper 4 gene cassette (Sense orientation)
Transcription of the Helianthus annuus Homeodomain-leucine zipper 4 (HaHB4) gene starts at the HaHB4 promoter and terminates at the Agrobacterium tumefaciens nopaline synthase gene terminator. The promoter activity is excepted to increase in response to abiotic stresses, ethylene and insect feeding, resulting in accumulations of HaHB4 transcript and HAHB4 protein.

HaHB4 nucleotide changes:
- Deletion of nucleotides 76 to 87
- A to G substitution at position 22 (codon change from AAA to AGA)
- G to A substitution at position 351 (silent point mutation - CAG to CAC)
- A to C substitution at position 401 (silent point mutation - GCA to GCC)
- T to C substitution at position 432 (codon change TTT to CTT)
- C to T substitution at position 551 (codon change CCT to CTT)

HB4 amino acid changes:
- Four amino acid deletion (residue numbers 7 to 10 - results in numbering change in residues)
- Lysine to Arginine substitution at position 22 (K22 -> R18)
- Phenylalanine to Leucine substitution at position 159 (F159 -> L155)
- Proline to Leucine substitution at position 175 (P175 -> L171)

Note:
- Southern blot, next generation sequencing and PCR analyses indicate that the plasmid backbone was absent in the genome.
- Southern blotting and sequencing confirmed the presence of a single, T-DNA insert.
- PCR analysis indicated that the transgene insert followed Mendelian inheritance.
- Bioinformatic analysis indicated that no new open read frames were created as a result of the T-DNA insertion.


Genetic elements introduced from PV-GMGT04
Agrobcterium tumefaciens 5-enolpyruvylshikimate-3-phosphate synthase (cp4-epsps) transcription is driven by the CaMV 35S Enhanced promoter and transcription terminates at the A. tumefaciens nopaline synthase terminator. The protein is localized to the chloroplast due to the inclusion of a Petunia hybrida chloroplast transit peptide 4 at the 5' end of the transcript. High levels of CP4-EPSPS transcript are expected due to the constitutive activity of CaMV 35S enhanced promoter.

Note:
- Southern blot and PCR analysis indicated that only a single transformation cassette containing the EPSPS coding sequence was integrated into the host genome.
- A deletion in the enhancer region of the CaMV 35S enhanced promoter occurred. However, the remainder of the E35S promoter was functional.
- Analysis also indicated that there was no integration of segments of the vector backbone, the GUS coding sequence or the CmoVb promoter.
- The expression of the EPSPS protein was confirmed by Western blotting, ELISA and EPSPS enzyme activity assay.

For more information, kindly refer to the parental LMO records.
LMO characteristics
Modified traits
Common use(s)
  • Food
  • Feed
Detection method(s)
Additional information
PAT protein (from HB4 soybean)
- the protein is expressed in the soybean forage and seeds at level comparable to or less than other crops expressing PAT
- highest levels of protein are detected in leaf and seed tissue

HB4 protein (from HB4 soybean)
- under normal conditions, the protein was difficult to detect in leaf and seed tissue (level below limit of detection)

EPSPS protein
- cp4-epsps coding sequence encodes a 455 amino acid protein and results in the synthesis of the full length and functional ~46kDa protein