When considering a medium for plant regeneration, there are a lot of considerations for optimizing and designing something that will be effective for what you’re working with. Different tissues, explants, even the stages are going to play a role in how you design your plant medium.

Extensive research has determined four steps for designing an effective plant medium: choosing the right medium for the different regeneration stages, choosing the right components, the right minor components, and the right antibiotics. Each of these steps requires careful consideration and optimization in order to produce an effective plant medium.

Having an appropriate culture medium is crucial for plant regeneration, which should be easily adapted for genetic transformation or micropropagation purposes.

Plant regeneration can be initiated to produce whole plants after genetic transformation. Plant regeneration can also be started from non-transformed tissues to propagate desirable traits already existing in the plants. Alternatively, callus formation can be initiated from non-transformed tissues in order to synthesize targeted secondary metabolites.

However, no matter what the goal is, whether synthesizing secondary metabolites or propagating transgenic plants, researchers commonly report that procedures developed for one species can seldom be applied to its relatives. This is true even for plants of the same genus.

Therefore, researchers should be careful to refine plant regeneration protocols at the species, subspecies, cultivar, or even donor tissue level.

This article describes the key components to develop a medium for in vitro plant regeneration, such as basal medium, carbon source, plant growth regulators, and other added minor components to improve the regeneration step. I also describe some considerations when working with antibiotics and some tips for preparing a plant regeneration medium.

Step 1 – Choosing the right media for different regeneration stages

Media composition affects the development of plant cells in the cultures by essentially influencing the plasticity (ability of the plants to adapt to and cope with changes in its environment) and totipotency (ability of plant cells through which an organ or plant can be regenerated) of the tissues used as explants.

Before going deeper into the plant regeneration media components, I should mention the three plant regeneration stages:

  • First stage – Plant tissue responds to the in vitro culture conditions and undergoes the dedifferentiation process. This is a process where differentiated cells become less specialized and return to an earlier cell state within the same lineage.
  • Second stage – Explants enter the induction phase, during which cells are identified to produce a shoot, root, or embryo.
  • Third stage Explants or derived callus (mass of undifferentiated plant cells) enter the realization stage, which results in the appearance of shoots, roots and embryos.

There are two categories of media in which the previous stages occur:

  • Induction medium: The first and second stages of plant regeneration are typically developed in induction medium. Callus formation is generally observed as a response to the induction medium, which also depends strongly on the medium composition. However, many factors, including explant source, temperature, growth conditions and donor plant variety also affect callus induction (Chodacka et al 2020).

  • Regeneration medium: Regeneration medium is most suitable for the third stage of plant regeneration. Researchers have shown regeneration medium has greater efficacy during the third stage of the plant regeneration process. In this medium, shoots, roots or embryos are regenerated directly from the explants or calli. Morphological characteristics of the newly formed structures can also be verified or investigated to see if abnormalities are present (Mangena, 2018).

The three stages of plant regeneration chart

Step 2. Choosing the key components for plant regeneration media

Based on several articles related with plant regeneration, I have found four major categories in which researchers perform optimizations: the basal medium, the carbon source (sugars), the plant growth regulators and minor added components.

Basal medium

A basal medium can be defined as a mixture of minerals in the form of salts to promote in vitro plant growth. They are designed by understanding the relevance of macro and micronutrients in the plant diet. Many researchers have reported the Murashige and Skoog (MS) basal medium in their plant regeneration protocols (Table 1). A similar salt mixture composition is commonly used in the induction and regeneration media.

However, some other mineral components have been reported to improve the callus growth (mainly embryogenic) in certain grasses and cereals; such as magnesium chloride and copper sulfate. Additionally, researchers have included some rapidly depleted substances in cultures such as potassium phosphate (Dalton, 2020). These components are always added as additional salts to the MS medium.

You might be thinking the preparation of an optimized medium with extra salts may appear complex, but in effect, it is easy to make. Basically, the additives can be simply combined in a stock solution and added to MS medium before autoclaving or filter sterilization.

I encourage you to take a look at our GoldBio article The Recipe for Plants: Strategies for Cell Culture Media Preparation to learn more about in vitro culture media.

Carbon source

Similar to MS basal mixture, sucrose is a commonly used carbon source in plant regeneration media. For both the induction medium and regeneration medium, sucrose is added in similar concentrations; usually between 2% to 3% or 20g/L and 30g/L, respectively.

Other authors have reported using maltose at 3% for cultures like Avena and Lillium (Dalton 2020). Although maltose is a common plant metabolite that is usually added to the media as a carbon source and osmoticum (causes osmotic stress), its role in transformation enhancement remains to be elucidated (Table 1). (Patel et al., 2013).

Plant growth regulators

A complete book would not be enough to describe all the attention needed to optimize the plant growth regulators (PGRs) in tissue culture. Because of their dose-effect relationship, each individual PGR requires its own specific tweaks. Indeed, PGRs are one of the most critical components in preparing a plant regeneration medium. Below is a brief explanation of the two main types of plant growth regulators: auxins and cytokinins.

Auxins

Auxin action has been reported in most, if not all, growth and developmental processes, in interactions with other hormonal signaling pathways, and even in the interaction with beneficial or pathogenic microorganisms and viruses (Weijers et al., 2018).

Here is a list of the most common auxins that are typically added to plant regeneration media:

Cytokinins

Cytokinins are plant hormones that influence numerous aspects of plant growth, development, and physiology; including cell division, chloroplast differentiation, delay of senescence and interaction with other organisms, including pathogens (Akhtar et al. 2020).

Among the cytokinins artificially added to a plant regeneration medium, we can find:

Plant cytokinins are divided into two main types:

  • Adenine-type cytokinins: BAP, 2-ip, kinetin, and zeatin.
  • Phenylurea-type cytokinins: Diphenylurea and TDZ are phenylurea-types, while TDZ is a potent cytokinin for plant tissue culture (Huang et al. 2010).

Approaches to Using Plant Growth Regulators in Medium

Most of these PGRs are added in the induction medium to promote the dedifferentiation process and callus formation. Later, PGRs are removed from the regeneration medium to facilitate natural plant development and recovery post-infection in Agrobacterium-mediated genetic transformation.

However, a few authors intentionally add PGRs to the regeneration medium as well. Therefore, we can find a large amount of information and protocols optimizing concentrations and types of PGRs for different cultivars and explants (Table 1).

Other PGRs like gibberellic acid and abscisic acid are used mainly to promote rooting or avoid tissue browning (explants become oxidated by the in vitro process).

List of plant growth regulators in either the auxin class or cytokinin class. These regulators are important factors in plant regeneration and plant function

Step 3. Choosing the correct minor components

For some recalcitrant monocot species, the addition of minor components is a common practice to improve regeneration efficiency (total number of regenerated shoots/embryos over the total explants). Some of the additives include:

  • Silver nitrate
  • Glutamine
  • Activated charcoal

For instance, activated charcoal enhanced the length of Lilium Leucanthum’s roots (a monocot) compared to the media without activated charcoal (Tang et al. 2010). This effect of activated charcoal on rooting could partly be attributed to the establishment of polarity in the darkening of the medium by charcoal and the adsorption of plant growth regulators and other organic supplements (Tang et al. 2010).

Group

Plant species

Basal medium

Carbon source

Plant growth regulators

Other components

Reference

Monocots

Glycine max

MS

Sucrose

BA

NA

Magena 2018

Indica rice

MS

Sucrose

Kin, NAA

NA

Saharan et al 2005

Lilium Leucanthum

MS

Sucrose

BAP, NAA

NA

Tang et al 2010

16 cereals

Modified MS

Maltose

BAP, IBA

proline

Dalton 2020



Dicots

Nigella damascena L.

MS

Sucrose

BAP, NAA

glycine

Chodacka et al 2020

Neolamarkia Cadamba

DCR

Sucrose

TDZ, NAA

NA

Huang et al 2010

Lycopersicon esculentum L. Mill.

MS

Sucrose

BAP, NAA

NA

Velvecha et al 2005

Physalis pruinosa

MS

Sucrose

BAP, NAA

NA

Swartwood and Eck 2019

Table 1. Description of components for plant regeneration media in a selected list of plants.

*MS: Murashige and Skoog medium

*DCR: Sugar pine medium

Step 4. Antibiotic selection

Initially, I mentioned plant regeneration is applicable for transformed and non-transformed explants. The addition of antibiotics to the plant regeneration medium must happen before the transformation process when working in vitro.

The aim of adding antibiotics is to arrest the Agrobacterium growth post-infection, favoring the plant cell recovery.

In this sense, an efficient and reproducible plant regeneration protocol is essential for the genetic manipulation of important crops.

The addition of antibiotics to the medium is a prerequisite when working with Agrobacterium-mediated transformation in order to arrest the bacterium growth post-infection.

However, the addition of antibiotics to the medium generally affects the callus responsiveness (transformed or not), commonly blocking or delaying the callus induction capacity (Dalton 2020, Bhau and Wakhlu, 2001).

Antibiotics also influence the explant survival, as was seen when significant amounts of callus cells appear in the controls without antibiotics compared to the explants infected with Agrobacterium (Mangena, 2018).

In soybean, MS media containing antibiotics likewise delays callus initiation. Mangena (2008) saw callus initiation was arrested for more than three weeks of culture. But later observed swelling and traces of slightly white-yellow friable callus (soft callus) on the explants. Similarly, in tomato, most of the transformed shoots did not undergo further growth and elongation in the medium containing antibiotics (Velvecha et al., 2005).

To overcome the challenges imposed by antibiotics in the medium, plant researchers evaluated different doses and types of antibiotics.

This evaluation led to standardized antibiotics being primarily favored such as:

It has also been found that a lower antibiotic concentration is preferred in order to arrest the Agrobacterium growth and at the same time promote the plant cell recovery.

For instance, better soybean shoot proliferation was achieved under aminoglycosides than the β-lactam antibiotics at a range between 50 and 500 mg/L. Also, 50 mg/L of kanamycin was effective in selecting transformed tomato cotyledon explants (Velvecha 2005).

Antibiotics help with cell recovery in plant transformation. Illustration about antibiotics in plant regeneration and transformation.

Key components to consider when preparing plant medium for plant regeneration: basal medium, major and minor components, and antibiotics. All need optimized to make an effective plant medium for regeneration.

Tips for plant regeneration

  • The type of explant strongly affects the response to a determined culture medium. For instance, immature embryos are usually cultured for callus induction in annual species such as Brachypodium distachyon, while mature embryos are used in species such as Oryza sativa. Additionally, meristems from Avena sativa shoot tips have been used for direct transformation (without a callus induction step).
  • An optimized medium for plant regeneration can reduce the time tissues are in the culture, reducing somaclonal variation since a decreased number of sub-cultures are required to produce callus.
  • Sub-culturing less than 0.5 g of callus to a 90 mm Petri-dish containing 25 ml medium improves callus growth rates. This amount probably represents the maximum plating density for callus in tissue culture (Dalton, 2020).
  • Using calli older than seven days can significantly reduce the transformation efficiency and affects the regeneration. It’s better to use callus no older than seven days.

4 tips for plant regeneration you can't live without.

Related products

GoldBio is committed to your plant research needs. Please visit our links to find related high-quality, affordable products

Plant culture media

Plant growth regulators

Antibiotics

References

Akhtar, S. S., Mekureyaw, M. F., Pandey, C., & Roitsch, T. (2019). Role of Cytokinins for Interactions of Plants With Microbial Pathogens and Pest Insects. Front Plant Sci, 10, 1777. https://doi.org/10.3389/fpls.2019.01777

Azadi, P., Chin, D. P., Kuroda, K., Khan, R. S., & Mii, M. (2010). Macro elements in inoculation and co-cultivation medium strongly affect the efficiency of Agrobacterium-mediated transformation in Lilium. Plant Cell, Tissue and Organ Culture (PCTOC), 101(2), 201-209. https://doi.org/10.1007/s11240-010-9677-9

Bhau, B.S., and Wakhlu, A.K. (2001). Effect of some antibiotics of the in vitro morphogenetic response from callus cultures of Coryphantha elephantides. Biologia Plantarum, 44(1), 19-24.

Bidabadi, S. S., & Jain, S. M. (2020). Cellular, Molecular, and Physiological Aspects of In Vitro Plant Regeneration. Plants (Basel), 9(6). https://doi.org/10.3390/plants9060702

Chodacka, M., Kadluczka, D., Lukasiewicz, A., Malec‑Pala, A., Baranski, R., Grzebelus, E. (2020). Effective callus induction and plant regeneration in callus and protoplast cultures of Nigella damascena L. Plant Cell, Tissue and Organ Culture (143):693–707. https://doi.org/10.1007/s11240-020-01953-9

Dalton, S. J. (2020). A reformulation of Murashige and Skoog medium (WPBS medium) improves embryogenesis, morphogenesis and transformation efficiency in temperate and tropical grasses and cereals. Plant Cell Tissue Organ Cult, 141(2), 257-273. https://doi.org/10.1007/s11240-020-01784-8

Huang, H., Wei, Y., Zhai, Y., Ouyang, K., Chen, X., & Bai, L. (2020). High frequency regeneration of plants via callus-mediated organogenesis from cotyledon and hypocotyl cultures in a multipurpose tropical tree (Neolamarkia Cadamba). Sci Rep, 10(1), 4558. https://doi.org/10.1038/s41598-020-61612-z

Joyce, P., Kuwahata, M., Turner, N., & Lakshmanan, P. (2010). Selection system and co-cultivation medium are important determinants of Agrobacterium-mediated transformation of sugarcane. Plant Cell Rep, 29(2), 173-183. https://doi.org/10.1007/s00299-009-0810-3

Mangena, P. (2019). The Role of Plant Genotype, Culture Medium and Agrobacterium on Soybean Plantlets Regeneration during Genetic Transformation. In Transgenic Crops – Emerging Trends and Future Perspectives. https://doi.org/10.5772/intechopen.78773

Patel, M., Dewey, R. E., & Qu, R. (2013). Enhancing Agrobacterium tumefaciens-mediated transformation efficiency of perennial ryegrass and rice using heat and high maltose treatments during bacterial infection. Plant Cell, Tissue and Organ Culture (PCTOC), 114(1), 19-29. https://doi.org/10.1007/s11240-013-0301-7

Saharan, V., Yadav, R., Yadav, N., & Chapagain, B. (2004). High frequency plant regeneration from desiccated calli of indica rice (Oryza Sativa l.). African Journal of Biotechnology, 3(5), 256-259. https://doi.org/10.5897/ajb2004.000-2047

Swartwood, K., & Eck, J. (2019). Development of plant regeneration and Agrobacterium tumefaciens mediated transformation methodology for Physalis pruinose. Plant Cell, Tissue and Organ Culture, (137), 465–472

Tang, Y.P., Liu, X.Q., Gituru, W, & Chen, L.Q. (2010). Callus Induction and Plant Regeneration from In Vitro Cultured Leaves, Petioles and Scales of Lilium Leucanthum (Baker) Baker. Biotechnology & Biotechnological Equipment, (24), 2071-2076. https: 10.2478/ V10133-010-0077-4

Velchevaa, M., Faltinb, Z., Flaishmanb, M., Eshdatb, Y., & Perl, A. (2004). A liquid culture system for Agrobacterium-mediated transformation of tomato (Lycopersicon esculentum L. Mill.). Plant Science, (168),121–130

Vermaa, S., Kumar-Dasb, A., Cingoza, G., Uslua, E., & Gurel, E. (2016). Influence of nutrient media on callus induction, somatic embryogenesis and plant regeneration in selected Turkish crocus species. Biotechnology Reports, (10), 66-74

Weijers, D., Nemhauser, J., & Yang, Z. (2018). Auxin: small molecule, big impact. eXtra Botany. https://academic.oup.com/jxb/article/69/2/133/4788745.