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 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 calli 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).
Step 2. Choosing the key components for plant regeneration media
Based on several articles related to 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 using 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 calli 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 that preparing an optimized medium with extra salts might be a little complex, but it is easy to do. 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 20 g/L and 30 g/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 medium 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:
- Indole-3-acetic acid (IAA)
- Indole-3-butyric acid (IBA)
- p-Chlorophenoxyacetic acid (4-CPA)
- Phenoxyacetic acid
- Picloram
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:
- 6-Benzylaminopurine (BAP)
- 6-(γ,γ-Dimethylallylamino)purine (2ip)
- Kinetin
- Zeatin
- Diphenylurea
- Thidiazuron (TDZ)
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 calli 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).
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).
Table 1. Description of components for plant regeneration media in a selected list of plants.
|
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 |
*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).
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.
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References
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