Plant tissue culture is a biotechnological technique for propagating plants.
To do this, plant fragments (explants) are transferred to an artificial growth medium supplemented with nutrients and hormones, on which they develop into complete plants. Tissue culture offers a range of advantages, such as consistent quality and performance, higher throughput, better uniformity and control of the transmission of plant diseases.
Therefore, this technique is an established commercial method of propagating high-value fruits and vegetables (e.g. bananas and berries), cosmetic, medicinal and ornamental plants (e.g. cannabis and orchids) and plants for forestry (e.g. bamboo, pine and spruce).
One important factor for successful plant development is the composition of the growth medium, which typically includes gelling agents to give support to the developing root system and facilitate the uptake of nutrients and water. The most commonly used gelling agents in plant tissue culture are agar/agarose, low-acyl gellan gum, carrageenan and alginate.
Xanthan gum is of interest as a texturant due to its high purity and quality, along with it being widely available on the market and, therefore, competitively priced. It has excellent thickening properties, but does not form solid gels as required in growth media when used by itself. However, gels can be produced by exploiting the synergistic interaction of xanthan gum with glucomannans or galactomannans.
Enhancing plant growth media with xanthan gum and other hydrocolloids
In 2024, Jungbunzlauer published a study in which the synergism between xanthan gum and other hydrocolloids (konjac gum, locust bean gum) was investigated with the aim to bring about specific benefits in gelling systems for growth media.1 The first part of the investigation focused on the impact of the novel gelling system on growth medium texture (rupture strength, elasticity).
The growth (rooting and shoot development) of a range of model plants was then determined for each gelling system used. This study shows how the incorporation of xanthan gum unlocks ways to control the textural properties of growth media, and how this can benefit different cultivars.
Agar and low-acyl gellan gum (LA GG) were selected as standard gelling agents. Xanthan gum was used in synergistic combinations with either locust bean gum or konjac gum. The combination of xanthan gum and galactomannans alone resulted in rather floppy gels, and agar had to be added to obtain better stability. Eventually, growth media were prepared with the following gelling systems:
- Agar
- LA GG
- Xanthan gum, locust bean gum and agar (XG-LBG-agar)
- Xanthan gum, konjac gum and agar (XG-KG-agar)
These different gelling systems were incorporated into Murashige Skoog plant nutrient medium and heated to 100°C under stirring. The solution was cooled down to 60°C under stirring and transferred to a single-cell measuring cup.
A texture analyser was used to perform a compression test to measure the rupture strength and the elasticity of the gels at defined intervals up to one month after preparation. The clarity and turbidity of the different gels were evaluated visually. The appearance of the gels is shown in Figure 1.
Figure 1: Appearance of media with different gelling systems: a) agar, b) LA GG, c) XG-LBG-agar, d) XG-KG-agar.
The growth medium with agar was turbid, firm and distinctly brittle. With LA GG, the gels were clear and firm. XG-LBG-agar and XG-KG-agar resulted in a firm yet flexible texture. XG-LBG-agar was crystal clear, while XG-KG-agar was largely clear.
Regarding texture, it was found that agar gels had the highest rupture strength, followed by the combinations with xanthan gum. LA GG had the lowest rupture strength. The most pronounced effect of the novel gelling systems was observed when looking at elasticity (Fig. 2).
Figure 2: Elasticity of media with different gelling systems over time, as determined by texture analysis (n=5).
The combinations with xanthan gum demonstrated the highest elasticity, whereas LA GG had the lowest elasticity.
The gels took some time to develop their elasticity, with the highest values being measured after 24 hours. Higher elasticity can make it easier to handle and transport plantlets on a growth medium, which may result in fewer plantlets being lost due to rupturing of the medium.
Growth and root development with XG-LBG-agar gelling system
The performance of plants on media prepared with different gelling systems was determined using representative ornamental, medicinal and forestry plants. These comprised Anthuriums, Echinacea, Helleborus and Fargesia. In pre-tests, XG-LBG-agar was selected as the best-performing test gelling system and compared to LA GG as a reference.
All trials were performed by Robotec PTC GmbH (Bremen, Germany). For each test cultivar, plants were grown in three identical SteriVent™ cups, which fit 36 individual plants each. Duration and parameters for evaluation were determined for each cultivar individually, depending on relevance and feasibility.
Anthurium has relatively simple demands of growth media and can thus be considered a positive control for investigating plant responses to different gelling systems. Evaluation was performed four weeks after cutting and transferring the explants to the growth medium. It was observed that all plants fulfilled the requirements for further processing – i.e. being rooted, with a shoot height of at least 3.5cm, a strong stem and at least three leaves.
However, the number of roots per SteriVent cup and the percentage of roots longer than 1cm varied between the gelling systems. Compared to the reference gelling system (LA GG), the use of XG-LBG-agar produced fewer roots per cup, but at the same time increased the proportion of roots longer than 1cm.
An optical evaluation of the canopy surface indicated that XG-LBG-agar may have a positive effect on leaf spread (Figure 3). Due to this positive outcome, it can be assumed that XG-LBG-agar is a generally plant-compatible gelling system.
Figure 3: Optical evaluation of shoot development and leaf spread of Anthurium cultivated with different gelling systems.
The development of Echinacea was evaluated after five weeks of cultivation. At this point, the majority of plants fulfilled the requirements for further processing, but the exact number of rejects varied depending on the gelling system. A plant was rejected if it had at least one of the following shortcomings: shoot height of less than 3cm, weak overall growth, fewer than three roots, lack of uniformity.
As shown in Figure 4, the percentage of rejected plants across all causes was greater when Echinacea was grown on LA GG than on XG-LBG-agar. The XG-LBG-agar gelling system therefore has the potential to improve economic efficiency by reducing the number of plant rejects.
Figure 4: Percentage of rejected Echinacea plants grown in different gelling systems, by cause of rejection.
The performance of Helleborus plants was evaluated after a cultivation period of eight weeks. For this cultivar, rooting under in-vitro conditions poses a particular challenge; its success was therefore a key indicator of gelling system performance.
On the medium with LA GG, only 58% of plants formed at least one root, whereas this value rose to 71% with XG-LBG-agar. An optical evaluation showed that above-ground biomass developed quite well regardless of the gelling system used.
As a challenging test for the growth media, a specific clone of Fargesia was selected which was known to lack root formation in vitro. In a commercial setting, these plants can be placed in soil once they reach a suitable stage of development, even though they have not formed roots.
The Fargesia plants were evaluated after eight weeks of cultivation. No root formation was observed with either gelling system. However, the plants did tend to develop better (larger shoot, greater leaf biomass) when grown on XG-LBG-agar than on LA GG, despite the lack of root formation.
Hydrocolloid combinations with xanthan gum
These results show that the combinations of hydrocolloids with xanthan gum complement the standard range of gelling agents and demonstrated distinct benefits in terms of texture – in particular, they resulted in firm, less brittle and more elastic gels than with agar or low-acyl gellan gum.
Higher elasticity reduces the susceptibility of the gel to breakage during handling and transport, thus keeping the growth medium intact during shipment of plantlets or if automated systems are in place.
In vitro plant cultivation trials proved the general suitability of the novel gelling system for a range of commercially relevant plants, highlighting plant-specific advantages, such as improved rooting of Echinacea. Combining xanthan gum with synergistic hydrocolloids thus offers a tool to fine-tune growth media properties to better meet plants’ needs and process requirements.
References
1. Jungbunzlauer: Facts: Xanthan Gum as a texturant in media for plant tissue culture.