Transgenic crops: how they are made
How transgenic crops are made: gene constructs, Agrobacterium and gene gun, selection, events, Bt cotton and GM mustard, biosafety rules and gene editing.
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A transgenic (genetically modified, GM) crop carries one or more genes inserted by laboratory methods instead of by crossing. Often the gene comes from an unrelated organism. Bt cotton carries a gene from the soil bacterium Bacillus thuringiensis that makes a protein toxic to bollworms. Herbicide-tolerant soybean carries a bacterial version of an enzyme that glyphosate cannot block. Golden Rice carries genes from maize and a bacterium that let the grain make β-carotene.
The first transgenic plant, a tobacco, was reported in 1983. The first commercial GM food, the Flavr Savr tomato, went on sale in 1994. GM crops now cover around 190 million hectares worldwide, mainly soybean, maize, cotton and canola. In India, Bt cotton, approved in 2002, is the only GM crop grown commercially at scale, and it now covers over 90% of the country's cotton area.
Step 1: The gene construct
The gene of interest is placed in a cassette with the sequences needed to make it work in a plant:
- a promoter to switch the gene on. CaMV 35S from cauliflower mosaic virus works in most tissues, and the maize ubiquitin promoter is used in cereals. Tissue-specific promoters target seed, root or green tissue.
- the coding sequence, often with its codons rewritten to suit plants. The cry genes in Bt crops were synthetically modified this way to raise expression.
- a terminator, such as nos, to end transcription.
- a selectable marker gene that lets transformed cells survive on a selective medium. Examples are nptII for kanamycin, hpt for hygromycin, and bar or pat for phosphinothricin.
The cassette is built in a bacterial plasmid and multiplied in E. coli.
Step 2: Delivering the DNA
Agrobacterium-mediated transformation
Agrobacterium tumefaciens causes crown gall disease. It naturally transfers a segment of its Ti (tumour-inducing) plasmid, the T-DNA, into the plant genome. The T-DNA is defined by two 25 base-pair border sequences, and virulence (vir) genes on the plasmid drive the transfer. Phenolic compounds such as acetosyringone, released by wounded plant cells, switch on the vir genes.
Scientists disarm the plasmid by removing the tumour genes, and place their own cassette between the borders, usually in a small binary vector. Explants such as leaf discs, cotyledons or immature embryos are co-cultivated with the bacteria. The method usually inserts few copies, with clean integration, and now works well in rice, maize, wheat and most dicots.
Direct gene transfer
- The gene gun (particle bombardment), developed by Sanford and colleagues, shoots DNA-coated gold or tungsten particles into cells. It works in any species but often inserts multiple, rearranged copies.
- Other direct methods are protoplast transformation by electroporation or PEG, and microinjection.
Steps 3 to 5: Selection, regeneration and confirmation
Only a small fraction of cells take up the gene. Explants are moved to a medium containing the selection agent, so only transformed cells grow. These cells are then regenerated into whole plants through tissue culture, giving the T0 generation.
Each T0 plant comes from an independent transformation event: the cassette has landed at its own random place in the genome. Events are confirmed and characterised by:
- PCR to detect the gene;
- Southern blot or digital PCR to count copies;
- RT-PCR, ELISA or Western blot to measure expression;
- bioassays, for example feeding bollworms on leaves.
How a single insert segregates
A T0 plant with one copy of the transgene is hemizygous (one copy on one chromosome), so it behaves like a heterozygote. When it is selfed, the T1 plants segregate 1 (two copies) : 2 (one copy) : 1 (no transgene). Testing for the transgene's presence therefore gives a 3 : 1 ratio. Out of 200 T1 seedlings, about 150 should test positive. A significant departure, such as 15 : 1, suggests two unlinked inserts. Breeders then pick homozygous T2 lines, which give 100% positive progeny.
Step 6: Event selection and backcrossing
Among dozens or hundreds of events, developers choose one with:
- a single, intact insert in a neutral part of the genome;
- stable expression over generations and environments;
- no unwanted effects on yield or quality.
That event is regulated and named. India's first Bt cotton, for example, used MON 531 (Cry1Ac). Bollgard II (2006) combined Cry1Ac and Cry2Ab. The event is then backcrossed into many locally adapted varieties and hybrids. This is why Indian farmers can choose hundreds of Bt cotton hybrids.
Regulation in India
GM crops in India are regulated under the Environment (Protection) Act, 1986, and the Rules of 1989. Three committees are involved:
- the Institutional Biosafety Committee (IBSC), at each research organisation;
- the Review Committee on Genetic Manipulation (RCGM), under the Department of Biotechnology, which approves contained research and trials;
- the Genetic Engineering Appraisal Committee (GEAC), in the environment ministry, which approves environmental release.
Biosafety assessment covers toxicity, allergenicity, gene flow, effects on non-target organisms and nutritional equivalence. The GEAC approved GM mustard DMH-11, based on the barnase–barstar male-sterility system, for environmental release in 2022. The case is still before the Supreme Court. Bt brinjal has been under a moratorium since 2010, though Bangladesh has grown it since 2014.
Genome editing
Genome editing with CRISPR–Cas9 and similar tools changes a plant's own genes precisely, often without leaving foreign DNA in the final plant. Edits are classified as SDN-1 (small changes without a template), SDN-2 (template-guided small edits) and SDN-3 (insertion of a gene). In 2022, India exempted SDN-1 and SDN-2 plants that carry no foreign DNA from the GM rules. In 2025 it released its first genome-edited rice varieties, DRR Dhan 100 (Kamala) and Pusa DST Rice 1.
Key points
- Transgenic crops carry genes inserted in the laboratory, often from unrelated species.
- A construct has a promoter (such as 35S), a coding sequence, a terminator and a selectable marker (such as nptII, hpt or bar).
- Agrobacterium transfers the T-DNA between 25 bp borders, with vir genes induced by acetosyringone. The gene gun works in any species but gives complex inserts.
- Transformed cells are selected, regenerated as T0 plants, confirmed by PCR, Southern blot and expression tests, and a single best event is chosen.
- A single-copy insert segregates 3 : 1 in T1.
- In India, GM crops are approved by IBSC, RCGM and GEAC. Bt cotton is approved; GM mustard DMH-11 was approved for release in 2022; Bt brinjal is under a moratorium. SDN-1 and SDN-2 edits are exempt.
What the exams ask
- Explain Agrobacterium-mediated transformation and the role of the Ti plasmid.
- Name selectable marker and reporter genes (gus, gfp).
- Define a transformation event and explain why several are produced.
- Describe India's biosafety regulatory structure.
- Distinguish transgenesis from genome editing.
Described in general terms: the kind of examination, not a past paper. Laisha never reproduces exam questions.
Questions people ask
Are GM foods safe to eat?
Major scientific reviews, including those of the US National Academies (2016), have found no evidence that approved GM crops are riskier to eat than their conventional counterparts. Each event is assessed separately, however. Agronomic issues such as pest resistance and gene flow also need managing, as the pink bollworm's resistance to Bt cotton has shown.
Why did pink bollworm overcome Bt cotton?
Pink bollworm feeds inside bolls and completes several generations a season. In India, refuges of non-Bt cotton were rarely planted, many hybrids flowered over a long period, and some seed was illegal or had low expression. Together these favoured resistant insects, which were widespread by about 2015.
What is a reporter gene?
A reporter gene produces an easily visible signal that shows where and when a promoter is active, or confirms transformation. Examples are gus, which turns tissue blue with a substrate, and gfp, which glows green under blue light.
Sources
- Slater, A., Scott, N. W. and Fowler, M. R. Plant Biotechnology: The Genetic Manipulation of Plants, 2nd edition. Oxford University Press, 2008.
- National Academies of Sciences, Engineering, and Medicine. Genetically Engineered Crops: Experiences and Prospects. National Academies Press, 2016.
- Gelvin, S. B. "Agrobacterium-mediated plant transformation: the biology behind the 'gene-jockeying' tool." Microbiology and Molecular Biology Reviews 67, 16–37 (2003).
- Kranthi, K. R. and Stone, G. D. "Long-term impacts of Bt cotton in India." Nature Plants 6, 188–196 (2020).
- Ministry of Environment, Forest and Climate Change. Rules for the Manufacture, Use, Import, Export and Storage of Hazardous Micro-organisms, Genetically Engineered Organisms or Cells, 1989.
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