Procedure

Basic steps for a gene stacking experiment in plants:

1. Choosing of the target traits and target plant

• Identification of the traits for improvements (e.g., drought tolerance, pest resistance, nutrient efficiency).
• Selection of corresponding genes/QTLs associated with each trait.

2. Designing of genetic construct

• Cloning of a single or multiple gene(s) into the multiple cloning site of the T-DNA or vector.
• Using of compatible promoters, terminators, and marker genes for each gene in the stack.

3. Plant Transformation

• Choosing of an appropriate transformation method:
  • Agrobacterium-mediated (commonly for dicots)
  • Biolistics (gene gun) (used for monocots like maize)
• Transformation into target crop.

4. Regeneration and Selection

• Growing of the transformants on selection media with antibiotics/herbicides.
• Regeneration of full plants from transformed tissues (callus → shoot → root).

5. Molecular Confirmation

• Confirmation of the presence and expression of all stacked genes using:
  • PCR / qPCR (for gene presence and copy number)
  • RT-PCR / qRT-PCR (for expression)
  • Southern blot (integration pattern)
  • Western blot (protein expression)

6. Phenotypic Screening

• Evaluation of the plants under controlled and field conditions for:
  • Trait expression (e.g., stress tolerance, pest resistance)
  • Agronomic performance

7. Generational Stability & Inheritance

• Growing of further generation of the transformant plants T1, T2, etc., for confirmation of gene expression and trait stability across generations.
• Performing Mendelian segregation analysis for each gene.

Here is a brief example of stacking two genes into a crop plant for enhanced drought and salinity tolerance via transformation using Agrobacterium tumefaciens:

Step 1: Selection of genes and preparation of recombinant Agrobacterium tumefaciens cells

1.1: Two genes were selected for two different traits. Gene 1 – overexpression of it will lead to enhanced drought tolerance. Gene 2 – overexpression of it will lead to enhanced salinity tolerance.
1.2: Using the isolated/synthesized genes that are commercially available for research use.
1.3: Subcloning of these genes into Ti-plasmid based plant expression vector under a single constitutive promoter. The genes were in a single ORF with a linker peptide in between.
1.4: Transformation of chemically competent Agrobacterium tumefaciens cells, and selection of true recombinant Agrobacterium tumefaciens cells. These recombinant Agrobacterium tumefaciens cells will be used for infecting the plant calli.

Step 2: Plant transformation and plant regeneration.

2.1: Plant callus were generated by the action of plant hormones (auxins and cytokinins)
2.2: The vir genes of the recombinant Agrobacterium tumefaciens cells were activated by phenolic compound acetosyringone, and were co-cultivated with the plant calli for infection in the dark for 48 hours.
2.3: The infected calli were regenerated into plants with the effect of plant hormones and were selected on the specific antibiotic.

Step 3: Molecular confirmation of the transgenic plants and growing in further generations

3.1: DNA and RNA were extracted from the putative transgenic plants and were confirmed by PCR/qPCR at the genomic/transcript level. Protein expression was checked by Western Blot.
3.2: After molecular confirmation and protein expression check, seeds were collected from the T0 plant and further plant generations were grown (T1, T2, etc.)
3.3: Molecular confirmation tests were carried out for the further generations.
3.4: Evaluation of the traits for improved drought and salinity tolerance.