You may agree that a good last name for Agrobacterium is not necessarily tumefaciens but “efficiency” because this is one of the parameters mostly pursued by researchers. Indeed, if you are working with plant transformation protocols, “transformation efficiency” should sound familiar as a close relative or a friend. However, similar to all relationships, to keep good connections you should know your friends very well. So, let me introduce what a transformation efficiency is, why it's so important, factors that impact Agrobacterium efficiency and ways to improve it.


What a transformation efficiency means?

Efficiency in a transformation protocol means the number of cells that were able to uptake foreign DNA molecules. So, researchers report “high” and “low” efficiency when working with transformation methods. Although an objective parameter for low and high efficiency is not well established, a low efficiency has been reported to be below 5% (Hayda et al., 2019), and high efficiency has been described over 30% up to 90% (Ozawa et al., 2009).


How is transformation efficiency calculated?

Calculating the transformation efficiency may vary between reports as researchers establish different experimental conditions. For example, when you are working with plants, one way transformation efficiency can be calculated is:

plant transformation efficiency formula version 1

Hence, a positive transgenic plant is defined as a plant that contains a gene that has been artificially inserted, and the insertion was verified by fluorescent methods or use of selectable markers.

An infected plant is defined as a plant treated by a transformation method such as protoplasts, biolistic or Agrobacterium-mediated. To learn more about plant transformation protocols take a look in our GoldBio’s article Deep Dive Into Plant Transformation Protocols: Protoplast-Mediated, Biolistic-Mediated and Agrobacterium-Mediated Gene Transfer.

However, you won’t always work with whole plants, and for specific studies like gene function some researchers work with detached vegetative tissues (i.e leaves). For instance, in species like Nicotiana benthamiana and lettuce with ~80% mesophyll cells, it is possible to achieve nearly 100% transient transformation efficiency in some regions of the leaf (Hwang et al., 2017). Here the transformation efficiency is calculated as:

plant transformation efficiency formula version 2


Is good transformation efficiency important when working with Agrobacterium cells?

You might be surprised, but in fact, the answer is “it depends.”

A high transformation efficiency could be required depending on the goals you are trying to achieve. Also, the aim of your experiment usually relates to a determined Agrobacterium method by using stable or transient transformation.

If the goal is regular cloning for breeding genetically modified (GM) crops, then a high transformation efficiency is a prerequisite, and stable transformation is primarily pursued.

In the stable transformation, the T- DNA is integrated into the plant genome and subsequently passed on to the next generation. Thus, stable transformation is a long process that often requires established tissue culture techniques to promote whole-plant growth from the transformed cells or tissues.

If the goal is specialized cloning for studies like gene function, protein function, and gene silencing (such as RNAi), high efficiency is not mandatory. These studies are mostly associated with transient transformation where T-DNA integration into the host genome is not required, and T-DNA expression usually lasts for a few days.

The idea in transient experiments is determining the gene or protein expression in short time. By consequence, the regeneration step is not necessary. Additionally, in a transient expression approach, high levels of expression in infected tissues can be achieved by delivering much higher numbers of T-DNA copies into plant cells than in a stable transformation protocol (Zhang et al., 2020).

Table 1. Guide of transformation efficiency based on applications.

Goal

Regular cloning

Specialized cloning

Type of studies

Transgenic plants

Gene function, protein function, gene silencing

Agro method

Usually stable transformation

Usually transient transformation

Efficiency

High

Low


What can go wrong with a lower transformation efficiency?

If you are using cells with a lower transformation efficiency, it can affect your downstream processes. One thing you should consider is that you will not get enough data to do a robust statistical analysis with lower efficiency. Also, you might not be able to provide strong conclusions based on few transformants. Furthermore, a low efficiency can hamper large-scale protocol reproducibility.

Table 2. Potential consequences of low transformation efficiency summary

Potential Consequences of Low Transformation Efficiency

Downstream processes

Data might not be robust enough for statistical analysis

Potentially incomplete conclusion due to too few transformants

Hinders large-scale protocol reproducibility

Consequently, a recommendation to overcome problems from lower efficiency is to determine what is/are the factor(s) causing lower efficiency.


How to improve the transformation efficiency

If you get a lower efficiency than you expected, keep calm and then start by analyzing the factors that influence the transformation efficiency. They include genotype, explant, strain, plasmid vectors, and culture conditions (Opabode, 2006).

factors impacting plant transformation efficiency infographic - agrobacterium competent cell transformation efficiency factors chart


Genotype

Dicotyledonous (plants producing two first leaves when germinating) are more susceptible to being transformed by Agrobacterium than monocotyledonous (plants producing only one first leave when they germinate). Still, within these groups, transformation efficiency varies among genotypes. It has been reported that the overall transformation frequency is higher in model cultivars (Cheng et al 2004).


Explant

Embryogenic callus derived from mature seeds and freshly isolated immature embryos have been reported to work well in monocotyledonous. For dicotyledonous, a larger spectrum of explants have been used including callus (undifferentiated mass of plant cells) from root sections (Nguyen et al. 2013) and meristematic tissues (Sabaddini et al., 2019).


Strain

A. tumefaciens strains are derivatives of the two wild-type virulent strains, C58 and Ach5. For instance, AGL1, EHA101, EHA105, and GV3101(pMP90) are derived from C58 and LBA4404 is derived from Ach5. Strains like GV3101(pMP90) from C58 and LBA4404 are regularly used for transient transformation of various plant species meanwhile a wider range of strains derived from both C58 and Ach5 have been used for stable transformation (Hwang et al., 2017). Previous works would guide the selection of the strain in related species to your target genotype.


Vectors

Agrobacterium vectors have evolved into various kinds, including binary, superbinary and ternary systems. Furthermore, using the combination of a standard binary vector in a supervirulent strain or a superbinary vector in a regular strain has been reported to influence the transformation efficiency. A super-Agrobacterium was reported to have reduced the time and labor required for transformations by approximately 72% than the previously developed strains (Nokana et al., 2019).


Culture-conditions

Some pretreatments in the explant have been reported to improve the efficiency. They include antinecrotic and osmotic treatments, desiccation, and temperature. Also, the addition of surfactants like Silwet L77 and Tween 20 improve the T-DNA delivery by aiding in the Agrobacterium attachment.

Additional factors have been evaluated, such as infection period, Agrobacterium density, the concentration of acetosyringone (an attracting-signal), and co-cultivation period (Utami et al., 2018). Furthermore, other factors such as donor plant health and stage of the donor material have also been reported by influencing the transformation efficiency (Hayda et al., 2019).


How do highly efficient agro cells help in the long run/ for plant transformation protocols?

To perform an effective and large-scale plant transformation study, highly efficient and simple genetic transformation methods for the studied organisms are crucial because they reduce cost and time. With more efficient Agrobacterium cells, you can get more transformants per experiment, favoring faster results.


Agrobacterium transformation efficiency improvement decision tree - chart showing how to decide how to improve transformation efficiency for Agrobacterium competent cells in plants


Related products

Test our GoldBio highly efficient Agrobacterium competent cells.



References

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Hayta, S., Smedley, M. A., Demir, S. U., Blundell, R., Hinchliffe, A., Atkinson, N., & Harwood, W. A. (2019). An efficient and reproducible Agrobacterium-mediated transformation method for hexaploid wheat (Triticum aestivum L.). Plant Methods, 15, 121. https://doi.org/10.1186/s13007-019-0503-z

Hwang, H. H., Yu, M., & Lai, E. M. (2017). Agrobacterium-mediated plant transformation: biology and applications. Arabidopsis Book, 15, e0186. https://doi.org/10.1199/tab.0186

Kirienko, D. R., Luo, A., & Sylvester, A. W. (2012). Reliable transient transformation of intact maize leaf cells for functional genomics and experimental study. Plant Physiol, 159(4), 1309-1318. https://doi.org/10.1104/pp.112.199737

Li, S., Cong, Y., Liu, Y., Wang, T., Shuai, Q., Chen, N., Gai, J., & Li, Y. (2017). Optimization of Agrobacterium-Mediated Transformation in Soybean. Front Plant Sci, 8, 246. https://doi.org/10.3389/fpls.2017.00246

Ma, R., Yu, Z., Cai, Q., Li, H., Dong, Y., Oksman-Caldentey, K. M., & Rischer, H. (2020). Agrobacterium-Mediated Genetic Transformation of the Medicinal Plant Veratrum dahuricum. Plants (Basel), 9(2). https://doi.org/10.3390/plants9020191

Martins, P. K., Ribeiro, A. P., Cunha, B., Kobayashi, A. K., & Molinari, H. B. C. (2015). A simple and highly efficient Agrobacterium-mediated transformation protocol for Setaria viridis. Biotechnol Rep (Amst), 6, 41-44. https://doi.org/10.1016/j.btre.2015.02.002

Nguyen, Q. V., Boo, K. H., Sun, H. J., Cao, D. V., Lee, D., Ko, S. H., Kang, S., Yoon, S., Kim, S. C., Park, S. P., Riu, K.-Z., & Lee, D.-S. (2013). Evaluation of factors influencing Agrobacterium-mediated spinach transformation and transformant selection by EGFP fluorescence under low-selective pressure. In Vitro Cellular & Developmental Biology - Plant, 49(5), 498-509. https://doi.org/10.1007/s11627-013-9534-8

Nonaka, S., Someya, T., Kadota, Y., Nakamura, K., & Ezura, H. (2019). Super-Agrobacterium ver. 4: Improving the Transformation Frequencies and Genetic Engineering Possibilities for Crop Plants. Front Plant Sci, 10, 1204. https://doi.org/10.3389/fpls.2019.01204

Opabode, J. (2006). Agrobacterium-mediated transformation of plants: Emerging factors that influence efficiency. Biotechnology Molecular Biology Review, 9.

Ozawa, K. (2009). Establishment of a high efficiency Agrobacterium-mediated transformation system of rice (Oryza sativa L.). Plant Sci, 176(4), 522-527. https://doi.org/10.1016/j.plantsci.2009.01.013

Sabbadini, S., Capriotti, L., Molesini, B., Pandolfini, T., Navacchi, O., Limera, C., Ricci, A., & Mezzetti, B. (2019). Comparison of regeneration capacity and Agrobacterium-mediated cell transformation efficiency of different cultivars and rootstocks of Vitis spp. via organogenesis. Sci Rep, 9(1), 582. https://doi.org/10.1038/s41598-018-37335-7

Utami, E. S. W., Hariyanto, S., & Manuhara, Y. S. W. (2018). Agrobacterium tumefaciens-mediated transformation of Dendrobium lasianthera J.J.Sm: An important medicinal orchid. J Genet Eng Biotechnol, 16(2), 703-709. https://doi.org/10.1016/j.jgeb.2018.02.002