Saving time is valuable when you are working with plant transformation protocols. However, you may know that some research goals can take years, meanwhile others just require a few weeks. In this article you will learn more about:

  • Stable vs. transient transformation protocols
  • How to differentiate them
  • Advantages and disadvantages of each type of transformation
  • When to apply them
  • How to know if a plant transformation protocol was successful.

What is the difference between stable vs. transient transformation?

Stable Plant Transformation

In a stable plant transformation, the foreign DNA is fully integrated into the host genome and expressed in later generations of the plant. This type of plant transformation is used for longer-term research of genes or production of a trait/compound on a large scale. For instance, a stable transformation project can take months, even years to be thoroughly developed.

Transient Plant Transformation

In contrast, a transient plant transformation allows temporary introduction or silencing of genes to determine their expression; thus, the foreign DNA is not integrated into the host cell. This method is mainly used for studying gene function or promoter behavior and protein function. It can also be used for gene silencing by expressing small interfering RNAs (siRNAs) and microRNAs (miRNAs) in plant tissues. In this case, a transient transformation can take days or weeks.

Using Agrobacterium in stable and transient transformations

In stable or transient transformation, Agrobacterium-mediated transfer is the preferred choice. To learn more about why Agrobacterium-mediated transformation remains popular, see our GoldBio’s article here.

Agrobacterium is a naturally occurring soil bacterium with the unique ability to transfer part of its DNA into plant cells. It is a phytopathogen (organism causing disease to plants) that causes the crown gall disease. Agrobacterium tumefaciens harbors the tumor-inducing (Ti) plasmid (inductor of galls on roots and crowns).

Inside this Ti plasmid, the transfer-DNA (T-DNA) region carries the oncogenes introduced in the plants to induce the tumor-like cell growth (e.g., auxin, cytokinin and opine genes). Close to this T-DNA region, there are the virulence genes (vir genes) required for T-DNA transfer. Usually, A. tumefaciens does not seriously harm plants unless galls occur in the root crown of young plants where they may become stunted and subject to wind breakage and drought stress.

Illustration of the plant Ti-Plasmid from agrobacterium - shows T-DNA and Virulence Genes

Figure 1. Components of Agrobacterium Ti-plasmid.

Regardless of the type of method you want to apply (stable or transient), you should consider the following requirements to utilize Agrobacterium and generate transgenic plants. First, use a nonpathogenic Agrobacterium strain (disarmed). The pathogenicity of the bacterium is eliminated by removing the oncogenes from the wild-type T-DNA. It avoids tumorigenicity, transforming normal plant cells into tumors). Thus, the vector is no longer harmful but is useful in delivering genes of interest to plants. Second, you should learn about DNA cloning techniques to introduce the gene of interest and selection markers into the T-DNA. Third, as the Ti plasmid is large and small in copy-number, other intermediated steps like E.coli use (this nice bacterium grows faster than Agrobacterium) are utilized to isolate and clone the Ti plasmid successfully.

Fortunately, Agrobacterium technology has improved significantly. Now researchers utilize a binary vector system. The T-DNA region is carried on a plasmid and the vir genes required for T-DNA transfer are located on the disarmed Ti-plasmid.

Agrobacterium binary vector illustration

Figure 2. Components of Agrobacterium binary vector.

Nowadays, T-DNA binary vectors have evolved, giving enormous versatility and flexibility to plant researchers. They include binary, superbinary and ternary vectors. The improvements in Agrobacterium vectors has also enabled an upsurge in transgenic plant production.

evolution of agrobacterium vectors, showing the single TI plasmid, binary vectors, superbinary vectors and trenary vectors

Altogether, among the differences between stable and transient transformation, we found 1) the goal to achieve in the experiment determines which method is more appropriate and 2) in the case of a transient system, as integration is not the primary purpose, a plant selectable marker is usually not required in the construct to be delivered to the host plant.

Advantages and disadvantages of stable and transient transformation

stable and transient plant transformation advantages and disadvantages

Techniques used in stable and transient transformation

Different explants can be used to apply stable or transient transformation. Below you can find a comparative table describing the different techniques used in plant transformation protocols.

Table 1. Comparison between stable transformation and transient transformation

Stable Transformation

Transient Transformation

Plant tissue


Reproductive inflorescences





Rooting explants and floral dipping

Agroinfiltration with a syringe or vacuum in roots, leaves and seedlings

Advantages of the techniques

Rooting explants

  • Cells are competent for regeneration and can be easily acquired in large quantities.
  • A relatively low percentage of transformants show polyploidy compared to leaves.
  • The method allows examination of transformation efficiencies of somatic cells.
  • The method facilitates determining the virulence of different A. tumefaciens strains.

Roots agroinfiltration

  • The binding ability of agrobacteria to the root tissue has been related to transformation efficiency.
  • Mutations hampering cellulose-associated genes can reduce the transient transformation efficiency in roots.

Floral dipping

  • Using this method avoids tissue culture steps and the resulting somaclonal variation.
  • A relatively short time is needed to acquire transformed progeny.

Leaves agroinfiltration

  • Some species are more susceptible to be transformed when leaves are used (e.g., Tobacco; good // Arabidopsis; no so good).
  • The strain C58C1 (harboring the octopine-type pTiB6S3DT-DNA and a pCH32 helper plasmid) is reported as the best in A. thaliana, lettuce, and tomato leaves (Wroblewski et al., 2005).
  • Suppress the plant immunity (e.g., with dexamethasone -DEX) can enhance the efficiency.
  • Leaf transient transformation has been employed for virus-induced gene silencing (VIGS).

Seedlings agroinfiltration

  • Techniques like FAST optimize the cell density (OD600=0.5) and concentration of Silwet L-77 (0.005%) without the need for vacuuming (Li et al. 2009).
  • EASI technique includes vacuum infiltration of young seedlings and a silencing suppressor (VirGN54D) within the same plasmid as the reporter gene (Mortensen et al., 2019).
  • C58C1(pTiB6S3DT-DNA, pCH32) is reported to achieve high transient transformation efficiency in A. thaliana seedlings.

Disadvantages of the techniques

In rooting explants, an established plant tissue culture system must exist for the genotype under study.

In floral dipping, the frequency of obtaining transformants in the next generation is highly variable.

The different tissues allow a specific range of application. For instance, root explants are mainly used only in root biology studies.


Months to years

Days to weeks

You can learn more about Transformation via Floral Dip here.

How to know if a stable transformation was successful?

Two approaches are used to detect the foreign DNA that has been integrated into the plant chromosome.

  • First, you can use the various antibiotic or herbicide resistance genes that have been inserted into the T-DNA region for transgenic plant selection. Usually, the neomycin phosphotransferase (NPTII) and hygromycin phosphotransferase (HPT) genes (conferring kanamycin and hygromycin resistance respectively), are the most widely used selectable marker gene systems in plants. Only the cells with successful DNA integration will be able to grow in a media with antibiotics added.
  • Furthermore, you can also use reporter genes to identify and quantify the transformed cells. The frequently used reporter gene products include ß-D-glucuronidase (GUS), both firefly and bacterial luciferases (LUC), and green fluorescent protein (GFP). These reporter genes work as fluorescent proteins. For GUS protein, blue staining is observed; meanwhile, LUC and GFP present a green color. Those tissues expressing the fluorescent proteins are an indicator of successful DNA integration.

How to know if a transient transformation was successful?

A gene of interest can be expressed separately with a GFP marker in one bicistronic vector (it allows the simultaneous expression of two proteins individually but from the same RNA transcript). The fluorescent GFP signals are used to identify and locate the cells that are successfully transformed by the vector. It also indicates the expression of the gene of interest.


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