TAM-66274-5 - Ultra-low gossypol cottonseed | BCH-LMO-SCBD-115357 | Living Modified Organism | Biosafety Clearing-House


Living Modified Organism (LMO)

Decisions on the LMO Risk Assessments  
last updated: 10 Jan 2020
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.
Ultra-low gossypol cottonseed
The cotton has been modified for ultra-low levels of the anti-nutrient, gossypol through the use of RNA interference (RNAi) targeting δ-cadinene synthase to disrupt gossypol biosynthesis. However, RNAi is constrained to the seed tissues and levels of gossypol are maintained in other tissues of the plant through seed specific expression of hairpin RNA. Thus, the modified cotton maintains gossypol protection from pests and diseases in other tissues, such as floral organs, foliage, roots and stem. Reduced levels of gossypol in the seed could allow the use of seed protein for food and feed. The modified cotton also contains the selectable marker Escherichia coli neomycin phosphotransferase II for kanamycin resistance and selection of transformants during tissue culturing.
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.
Gossypium hircutum cultivar Coker 312
Characteristics of the modification process
  • Agrobacterium-mediated DNA transfer
Some of these genetic elements may be present as fragments or truncated forms. Please see notes below, where applicable.
  • BCH-GENE-SCBD-100271-5 Octopine Synthase Gene Terminator | Agrobacterium tumefaciens (Agrobacterium)
  • BCH-GENE-SCBD-115354-3 δ-cadinene synthase dsRNA | Gossypium hirsutum (Cotton)
    Double-stranded RNA
  • BCH-GENE-SCBD-103123-6 Pyruvate orthophosphate dikinase, Intron 3 | Flaveria trinervia (Clustered Yellowtops, speedyweed, flaveria, yellow twinstem)
  • BCH-GENE-SCBD-115355-3 5' untranslated region of α-globulin B | Gossypium hirsutum (Cotton)
  • BCH-GENE-SCBD-100270-6 Nopaline Synthase Gene Promoter | Agrobacterium tumefaciens (Agrobacterium)
  • BCH-GENE-SCBD-15171-5 Nopaline Synthase Gene | Agrobacterium tumefaciens (Agrobacterium)
    Protein coding sequence | Selectable marker genes and reporter genes
  • BCH-GENE-SCBD-15001-5 Neomycin Phosphotransferase II | Escherichia coli (ECOLX)
    Protein coding sequence | Resistance to antibiotics (Kanamycin)
  • BCH-GENE-SCBD-100269-8 Nopaline Synthase Gene Terminator | Agrobacterium tumefaciens (Agrobacterium)
  • BCH-GENE-SCBD-115356-1 bleomycin binding protein | Escherichia coli (ECOLX)
    Protein coding sequence | Resistance to antibiotics,Resistance to bleomycin
  • BCH-GENE-SCBD-101415-9 Ti plasmid left border repeat | Agrobacterium tumefaciens (Agrobacterium)
    Plasmid vector
RNA interference cassette
Transcription of the RNA interference (RNAi)-triggering hairpin RNA (hpRNA) is under the control of the Gossypium hirsutum 5' untranslated region of α-globulin B gene and the Agrobacterium tumefaciens octopine synthase terminator. The transcript consists of a 604 basepair segment of the δ-cadinene synthase, a Flaveria trinervia pyruvate orthophosphate dikinase (pdk) intron and the reserve complement of the 604 basepair segment of the δ-cadinene synthase. The pdk intron acts as a spacer, allowing for looping such that the two δ-cadinene synthase (dCS) segments can basepair and form a double stranded section after transcription. Due to the processing of the hpRNA during the RNAi response, no protein is expected to be produced (kindly refer to the section "How the expression of the gene(s) was affected"). The production of the hpRNA is constrained to seed-specific tissues due to the specificity of the 5' untranslated region of α-globulin B gene. Thus, RNAi of  δ-cadinene synthase is not expected to be observed in other tissues of the plant.

Notes regarding the RNAi trigger sequence
Cotton species contain two subfamilies dCS genes: CAD-1A and CAD-1C, the latter containing multiple genes wihtin the genome. Thus, the sequence for dCS was chosen such that it shares 80.9 to 99.8% homology to several published sequences of dCS genes in diploid (Gossypium arboreum) and tetraploid (G. hirsutum) cottons. Thus, allowing the RNAi-mediated targeting of all members of the dCS gene family.

Selectable marker
Escherichia coli neomycin phosphotransferase II is under the control of the A. tumefaciens nopaline synthase (nos) promoter and terminator. The transcript is expected to include the following intervening sequences, which are not expected to form functional proteins: a partial sequence from the 5' end of nos; a partial sequence of the 5' end of the bleomycin resistance gene; and a partial sequence from the 3' end of nos.

Notes regrading T-DNA insertion:
- Southern blot analysis revealed that a single insert of the T-DNA is present in the genome
- HE-TAIL PCR showed that the right border was not integrated into the cotton genome. However, 7 nucleotides from the left border were integrated.
- No genetic elements from the vector backbone were integrated
- T-DNA integration occurred in the an intron of a putative α-hydrolase gene. QPCR analysis demostrated that there was no impact on mRNA expression compared to non-transgenic cotton.
- Southern blot analysis demonstrated inheritance of the insert follows Mendelian inheritance.
LMO characteristics
Delta-cadinene synthase
Upon transcription of the hpRNA in seed tissues, the cell recognizes the double stranded nature of the RNA transcript. Thus, an RNAi response is triggered. Host DICER protein binds the hpRNA, fragmenting it into 21-24 nucleotide small interfering RNAs (siRNA), which are then bound by ARGONAUTE proteins that further unwind the siRNA and form the RNA-induced silencing complex (RISC). Now activated, RISC acts as an endonuclease, degrading transcripts with a complementary sequence to the siRNA. Thus, the host cell targets its own endogenous δ-cadinene synthase mRNA, silencing expression of the gene and preventing gossypol biosynthesis.
  • Food
  • Feed
  • Fiber/textile
Detection method(s)
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