Photoautotrophic micropropagation – an overview

When you grow plants using tissue culture, it is common to encounter culture media with sugar. This sugar supplies carbon to your plants for growth and development. However, did you ever wonder about how a plant could grow in a sugar-free medium?

Plants can grow in three ways under in vitro systems. These are heterotrophic, photomixotrophic, and photoautotrophic growths. The most common growth mode is heterotrophic growth, where growth solely depends on the sugar present in the medium. Then there is photomixotrophic growth where plant development depends on the sugar in the medium as well as on the photosynthetic ability of the plant. Lastly, there is photoautotrophic growth, whereby plants make their own food using light as an energy source. Therefore, considering the principle of photoautotrophic growth, the method of 'photoautotrophic micropropagation' has been developed.

What is photoautotrophic micropropagation?

Photoautotrophic micropropagation ('PAM') is an in vitro propagation method where you can develop plantlets in a sugar-free medium containing only inorganic components. The media you use for this technique does not contain supplementary organic components such as vitamins and plant hormones. Also, this media does not include gelling agents, instead, you can use other materials with high air porosity such as rockwool, perlite, etc.

So, you might be wondering what leads to plant growth under photoautotrophic micropropagation? The answer is that plant growth in the PAM depends entirely on photosynthesis as well as the plant's ability to take in inorganic nutrients.

Research has revealed that chlorophyll cultures (such as leaf explants, somatic embryos, etc.) have high photosynthetic capacity when grown in vitro with a good source of carbon dioxide. Therefore, in the PAM system, photosynthesis is promoted by providing favorable environmental conditions for plants to synthesize primary and secondary metabolites necessary for their growth and development.

In addition to the technique of photoautotrophic micropropagation, there are some more methods of tissue culture to obtain healthy in vitro plants. You can read more about them in our article "7 methods of plant tissue culture".

What are the growth stages in PAM?

A PAM system consists of three stages. They are:

• Stage 0 - Stock plant selection and preparation.
• Stage 1 - Initiation/establishment of aseptic cultures. This step can be carried out under heterotrophic/photomixotrophic conditions by culturing meristematic tissue. Once the cultures develop photosynthetic organs (e.g., leaves), they are then transferred to PAM conditions.
• Stage 2 - Multiplication/rooting. Here multiplication and rooting, both are combined by reproducing photosynthetically active nodal cuttings and used as explants.

What environmental conditions do you need for PAM?

Controlled environmental conditions are key for the growth of healthy plants. When you grow plants in field conditions, the important factors are the availability of water and nutrients, temperature as well as salinity. Their absence can generate stress for the plants. However, under in vitro conditions, plants experience a controlled environment and hence, no stress. When you use the PAM system, you need to be careful with factors influencing the process of photosynthesis.

The important factors in a PAM system to promote photosynthesis and enhance plant growth are:

• Carbon dioxide concentration;
• Light intensity;
• Relative humidity; and
• Ventilation rate in the culture vessels.

Thus, you need to monitor these parameters and efficiently adjust them. In general, the physical conditions under PAM should be:

• Low relative humidity in the vessel;
• High carbon dioxide concentration during the photoperiod;
• High light intensity in the vessel and PPF ('Photosynthetic Photon Flux' is the amount of light energy used by plants during photosynthesis); and
• High vessel ventilation rate.

The levels of these factors vary with different plant species and their physio-ecological state. Among these conditions, carbon dioxide supply and ventilation rate are the most challenging tasks. So, how do you achieve them?

System configuration

Researchers have identified two effective ventilation systems for carbon dioxide supply. They are:

• Natural ventilation: This system is mainly used in small vessels. This functions on the basis of natural exchange of gases, through one or two gas permeable filters on the vessel lid.
• Forced ventilation: Here carbon dioxide enriched air is pumped into the large vessels through a gas-permeable filter disk. Therefore, you can easily adjust the ventilation rate to the necessary photosynthesis rates at different stages of plant growth.

You can choose the ventilation system on the basis of the scope of your micropropagation experiment as well as the available explants.

Advantages

PAM is a unique approach to producing in vitro quality plantlets. But, why should you use this technique?

Here are some of the advantages offered by this technique in comparison to conventional micropropagation:

• You can obtain plants that are vigorous, healthy and have an improved physiological state without morphological disorders.
• This method leads to reduction in microbial contaminations as the main source of contamination (sugar) is not present.
• The in vitro plantlets show higher survival rates in greenhouse and field conditions.
• There is no requirement of a separate acclimatization phase.
• There is an easier automation of the culture system and scaling up as you can use large culture vessels/rooms with flexibile designs.
• In some plants, there is an increase in expression of genes that are associated with photosynthesis efficiency.
• The possibility of mutations is lower as there is little or no use of plant growth regulators in culture media.

Disadvantages

Major disadvantages of photoautotrophic micropropagation are:

• It is crucial to have strong knowledge of the in vitro and ex vitro environment, physiology of the plantlets, and the physical characteristics of the culture vessels for this method to be successful.
• There may be an increase in costs as you need to use instruments which facilitate high concentration of carbon dioxide in culture vessels.
• Its application in plants with C4 or CAM photosynthesis pathway is limited. C4 and CAM photosynthesis pathways are present in plants (such as sugarcane, cacti, pineapple, etc.) that are best adapted to hot and dry environments. These processes ensure efficient production of energy in plants that grow in extreme environments. We will discuss in detail about the different photosynthesis pathways in upcoming articles.

What are the applications of PAM?

Some important applications of photoautotrophic micropropagation are:

• This technique is useful for biochemical production from medicinal plants. For instance, 'hyperforin' from St. John’s wort plant.
• In somatic embryogenesis, PAM can enhance embryo germination and their conversion to plantlets.
• PAM serves as a research tool for testing phenotypes of genetically engineered plantlets under aseptic and controlled environment conditions.
• This method is an effective alternative for producing plants of interest in the horticulture industry.

We hope this article gives you a better understanding of photoautotrophic micropropagation. For more informational posts on different aspects of plant tissue culture, keep checking this space!

By Valeria Franco Franklin | 9-November-2021

About the author

Valeria Franco is from Colombia, the land of orchids. She is a focused and passionate biologist who specializes in biotechnology and molecular biology. Valeria has prior laboratory and research experience. She is presently employed as a content creator at Lab Associates and is always looking for new challenges. Valeria is enthusiastic in plant science themes and reading as a tool for lifelong learning. Her hobbies include studying foreign languages, traveling, and archery.

References

• Xiao, Y., Niu, G. & Kozai, T. (2011). Development and application of photoautotrophic micropropagation plant system. Plant Cell Tiss Organ Cult 105, 149–158. https://doi.org/10.1007/s11240-010-9863-9
• Kubota, C. (2001). Concepts and Background of Photoautotrophic Micropropagation. Molecular Breeding of Woody Plants, Proceedings of the International Wood Biotechnology Symposium (IWBS), 325–334. doi:10.1016/s0921-0423(01)80089-7
• Nguyen, Q. T., Xiao, Y., & Kozai, T. (2020). Photoautotrophic micropropagation. Plant Factory, 333–346. doi:10.1016/b978-0-12-816691-8.00023-6 
• Kozai, T., & Xiao, Y. (n.d.). A Commercialized Photoautotrophic Micropropagation System. Focus on Biotechnology, 355–371. doi:10.1007/978-1-4020-3694-1_19 
• Ashrafzadeh, S., & Leung, D. W. M. (2021). Photoautotrophic micropropagation system (PAM): a novel approach deserving increased uptake for commercial plant production. Vegetos, 34(1), 13–18. doi:10.1007/s42535-020-00182-x 
• Zarei, A., Behdarvandi, B., Tavakouli Dinani, E., & Maccarone, J. (2021). Cannabis sativa L. photoautotrophic micropropagation: a powerful tool for industrial scale in vitro propagation. In Vitro Cellular & Developmental Biology - Plant. doi:10.1007/s11627-021-10167-3 
• Teixeira Da Silva, J.A., Giang, D.D.T. & Tanaka, M. (2006). Photoautotrophic micropropagation of Spathiphyllum . Photosynthetica 44, 53–61. https://doi.org/10.1007/s11099-005-0158-z