Overview

As an important model plant, Nicotiana benthamiana has the advantages of short culture cycle and easy cultivation, and can complete the post-translational modification of foreign proteins in its body, so it is more suitable for functional verification of heterologous genes than E. coil and yeast (Reed & Osbourn, 2018). At the same time, with the maturity of Agrobacterium-mediated transient gene transformation system, it has become more convenient to express foreign proteins in Nicotiana benthamiana transiently, quickly and in large quantities for functional research (Sheludko et al., 2007). In our project, we used Nicotiana benthamiana as our chassis organism and we also encountered various problems in our experiments. Based on our own experimental experience and data, we have compiled some common problems and solutions that teams performing tobacco transient expression in iGEM competitions may encounter.

Troubleshooting

Transient expression experiments generally select 4-week-old tobacco plants with leaves of appropriate size, preferably dark green and thick leaves. In order to obtain the best experimental materials, the conditions such as light, temperature, humidity, and watering frequency need to be controlled during the planting process. During the injection process, we should also pay attention to controlling the concentration of the bacterial solution and operate carefully to avoid damaging the leaves and ultimately affecting the experimental results. We've listed five common problems and their solutions below.

Section Ⅰ: Nicotiana Benthamiana Cultivation

Trouble 1: Tobacco seeds are difficult to germinate when sown in the soil.
Analysis: The seeds were planted too deep and there was not enough sunlight to stimulate germination; the soil moisture did not reach the conditions for seed germination.
Solution: Tobacco seeds should be sown shallowly, and a layer of plastic film is usually covered on the soil surface to keep the soil moist. In order to improve the survival rate, the surface-sterilized tobacco seeds can be planted on 1/2 MS culture medium. After growing in the incubator for one week, the seedlings can be moved into the soil.

Fig. 1 Tobacco seedlings germinated on 1/2 MS medium.

Trouble 2: The buds from tobacco seeds show a phenotype of tall stems and small leaves.

Fig. 2 Germinated tobacco seedlings show a phenotype of tall stems and small leaves.

Analysis: Tall stems and small leaves may be caused by short light hours, dim light, or excessive watering.
Solution: If conditions permit, try to grow tobacco in a light incubator and set the parameters: temperature (20-25°C), light (10,000–15,000 lux), and photoperiod (16h light/8h dark). At the same time, follow the principle of moderate watering to keep it moist but avoid waterlogging.

Trouble 3: White fuzz grows on the surface of the soil where tobacco is grown, affecting the growth of the plants

Fig. 3 The soil surface of tobacco planting has a layer of white fuzz.

Analysis: White fuzz is often the result of mold growth. Keeping the soil surface too moist provides an ideal environment for mold to grow.
Solution: Sterilize the soil before use; spray the soil surface with fungicides that are harmless to plants; control the frequency of watering, and do not over-water.

Section Ⅱ: Agrobacterium-mediated transient expression in tobacco

leaves

Trouble 4: It is difficult to inject the Agrobacterium solution into the experiment using a syringe.

Fig. 4 Using a syringe to inject Agrobacterium solution into tobacco leaves.

Analysis: The selected leaves are too old or the injection site is located in the veins of the leaves, which will result in difficult injection.
Solution: Select smooth and tender leaves or choose to inject during the day when the stomata of the leaves are open rather than at night when the stomata are closed. You can also use a needle to pierce a small hole before injecting, which can make the injection easier.

Fig. 5 Schematic diagram of tobacco in ideal conditions for experiment and correct injection operation.

Trouble 5: Tobacco leaves wilt after injection.

Fig. 6 After the bacterial solution was injected, the leaves became wilted.

Analysis: The Agrobacterium solution injected into the body will cause certain damage to the leaves. If too much bacterial solution remains on the surface of the leaves, it will also cause the leaves to wilt.
Solution: Appropriately reduce the concentration of the injected bacterial solution, and ensure that a single multi-dose injection is used instead of multiple small-dose injections to reduce leaf damage caused by more trauma sites. After the injection, be sure to discard the excess bacterial solution on the leaves.

Summary

A negative result in an experiment may be caused by multiple factors. Our troubleshooting may not be comprehensive, but we hope you can get some inspiration to improve your experiments.

References

[1] Reed, J., & Osbourn, A. (2018). Engineering terpenoid production through transient expression in Nicotiana benthamiana. Plant cell reports, 37(10), 1431-1441.
[2] Sheludko, Y., Sindarovska, Y., Gerasymenko, I., et al. (2007). Comparison of several Nicotiana species as hosts for high‐scale Agrobacterium‐mediated transient expression. Biotechnology and Bioengineering, 96(3), 608-614.