The efficient micropropagation protocol of Linnaea borealis var. borealis was established previously using the method of stimulation of new buds from pre-existing meristems. The influence of the type of the plant explant (single, double and triple shoots), hormonal supplementation in the medium, and culture system on shoot multiplication were estimated (Thiem et al. 2021). On this basis, the most appropriate type of explant (double shoots), a medium variant (BAP 1.0 mg/l + IAA 1.0 mg/l + GA3 1.0 mg/l and controls) as well as a culture system (a temporary immersion bioreactor), which combine the advantages of growing shoots on a solid or in liquid medium were selected in order to increase the scale of shoot cultivation.
In this study, the shoots of L. borealis were propagated by stimulating the division of meristematic cells located in the nodal parts of the stem and in the apical part of the shoot, using double shoots as explants.
All explants placed in the liquid medium on the rotary shaker gave a response, which proves the high morphogenic potential of the plant, regardless of the medium variant. However, the supplementation of the nutrient solution in PGRs significantly influenced the number of new shoots and the growth of fresh shoot mass. The highest results of growth parameters (more than 18 shoots per explant and more than 2000% biomass increase) for this type of culture system were obtained after 6 weeks for shoots agitated in a medium enriched with cytokinin and auxin (18.32 ± 0.52 and 2225.46 ± 91.81 respectively) as well as cytokinin auxin and gibberellin (18.26 ± 0.35 and 2184.95 ± 98.12 respectively) (Table 1).
Table 1
The influence of hormonal supplementation of MS medium on L. borealis shoot biomass growth parameters using a system of shoots agitated in a liquid medium
Medium Variant
|
Percentage
of Response
|
Multiplication
Rate
|
Fresh Biomass Growth Ratio (Mean ± SE)
|
Hyper-hydricity
|
MS
|
100%
|
4.32 ± 0.08c
|
541.48 ± 62.21c
|
NO (0%)
|
MS + BAP
|
100%
|
12.21 ± 0.13b
|
1233.26 ± 73.41b
|
NO (0%)
|
MS + BAP + IAA
|
100%
|
18.32 ± 0.52a
|
2225.46 ± 91.81a
|
NO (0%)
|
MS + BAP + IAA + GA3
|
100%
|
18.26 ± 0.35a
|
2184.95 ± 98.12a
|
NO (0%)
|
MS – Murashige & Skoog medium; BAP – 6-benzylaminopurine; IAA – indole-3-acetic acid; GA3 – gibberellic acid; Mean values within a column with the same letter are not significantly different at P = 0.05 |
(Duncan’s Multiple Range Test) |
Despite the use of liquid media, the shoots were characterized by the correct morphology and the vitrified shoots were not observed (Fig. 1,2), which is also due to the correctly selected volume of the medium in relation to the size of the culture vessel. The explants were not completely immersed in the medium and only the liquid rinsed them in the rhythm of shaking. Farahani and Majd (2012) concluded that the lack of oxygen in the liquid media containing small explants is the major limiting factor to growth.
An interesting article comparing the stationary and shaking system of running a shoot culture with the use of a liquid medium is the work on the medicinal species Salvia officinalis L. The use of the agitated culture system in vitro was not recommended in the case of sage cultivation. Shoots obtained by this method were verified and necrotic, while those growing in the stationary system on the medium with the addition of agar had the correct morphology (Grzegorczyk et al. 2008). Another article describing the comparison of the stationary and agitated system of shoot cultures is the work on the species Eryngium alpinum L. As shown by the results, plants propagated with the use of agar-solidified medium were characterized by correct morphology and intense green color, which distinguishes them from plants from the agitated system, in which verified shoots could be observed. The cultures conducted in the stationary system were also characterized by a greater number of new shoots compared to the agitated system. In the case of this species, it was probably an improper ratio of the volume of the medium to the culture vessel (Kikowska et al. 2020). On the other hand, the work of Merhotra team shows the many advantages of using the in vitro culture system in a liquid medium in the process of micro-propagation. The author states that the use of a liquid medium is associated with better uptake of nutrients and phytohormones by plant tissues during cultivation, which is responsible for improving the condition of the plant and generating proper development. Another advantage of using a liquid medium is the reduction of apical dominance, which affects the development of side buds and a greater increase in biomass (Merhotra et al. 2007).
As the research shows, the supplementation of the nutrient medium with phytohormones has a significant impact on the proper development of shoots in in vitro cultures. Experiments carried out on 3 subspecies of Hypericum perforatum L. using liquid MS medium in an agitated system indicated that the greatest increase in biomass could be observed when the ratio of cytokinins to auxins was 1: 1. Morphology of growing shoots in in vitro cultures changed with increasing concentration of phytohormones: at the lowest concentration formed normal, green shoots were observed; while at the highest concentration the shoots were underdeveloped and a developing callus was observed at their base (Kwiecień et al. 2018).
When analyzing the influence of nutrient supplementation, it can be concluded that full supplementation with BAP + IAA + GA3 gives the best results both in terms of multiplied shoots, length of explants and biomass growth of E. alpinum (Kikowska et al. 2020). The literature shows that cytokinins such as BAP favorably influence the development of side buds in shoot cultures of many plant species e.g. Rubus chamaemorus L. (Thiem 2001), Lychnis flos-cuculi L. (Maliński et al. 2019), Plantago media L. (Budzianowska et al. 2019), Chaenomeles japonica L. (Kikowska et al. 2019) while gibberellin GA3 lengthens the internode parts of shoots (Zhang et al. 2016; dos Santos et al. 2017).
All shoots grown in the RITA® bioreactors, regardless of the type of supplementation, were characterized by the correct morphology and viability. The presence of hyperhydricity and callus was not confirmed in any of the treatments (Fig. 3).
The supplementation of the nutrient solution in PGRs significantly influenced the number of new shoots and the enhancement of fresh shoot mass. The highest results of growth parameters (more than 10 shoots per explant and more than 600% biomass increase) for this type of culture system were obtained for shoots temporary immersed in a medium enriched with cytokinin, auxin and gibberellin (10.32 ± 0.43 and 681.35 ± 35.45, respectively) (Table 2).
Table 2
The influence of hormonal supplementation of MS medium on L. borealis shoot biomass growth parameters using RITA® system of shoots immersed in a liquid medium
Medium Variant
|
Percentage
of Response
|
Multiplication Rate
|
Fresh Biomass Growth Ratio (Mean ± SE)
|
Hyper-hydricity
|
MS
|
80%
|
3.52 ± 0.18d
|
214.49 ± 37.52c
|
NO (0%)
|
MS + BAP
|
100%
|
5.21 ± 0.12c
|
241.77 ± 15.54c
|
NO (0%)
|
MS + BAP + IAA
|
100%
|
8.62 ± 0.22b
|
483.98 ± 67.40b
|
NO (0%)
|
MS + BAP + IAA + GA3
|
100%
|
8.54 ± 0.46b
|
451.87 ± 57.99b
|
NO (0%)
|
MS + BAP + IAA + GA3 + AS
|
100%
|
10.32 ± 0.43a
|
681.35 ± 35.45a
|
NO (0%)
|
MS – Murashige & Skoog medium; BAP – 6-benzylaminopurine; IAA – indole-3-acetic acid; GA3 – gibberellic acid; AS – adenine sulfate. Mean values within a column with the same letter are not significantly different at P = 0.05 (Duncan’s Multiple Range Test) |
The shoots have grown in 100 and 150 ml of liquid medium were characterized by viability, correct morphology and the highest growth parameters (multiplication rate 10.23 ± 0.24 and 12.92 ± 0.33, respectively; biomass growth ratio 849.83 ± 76.21 and 894.31 ± 18.02, respectively). On the other hand, the shoots immersed in 80 ml of the nutrient solution were drying out, therefore the fresh growth biomass ratio decreased during the culture. The shoots immersed in 200 ml of the medium were characterized by overgrowth and hyperhydricity, and therefore, more than as a result of the multiplication of shoots, the ratio of fresh growth biomass was relatively high (Table 3).
Table 3
The influence of medium volume in bioreactor vessels on L. borealis shoot biomass growth parameters using RITA® system of shoots immersed in a liquid medium
Medium Volume
|
Percentage
of Response
|
Multiplication
Rate
|
Fresh Biomass Growth Ratio (Mean ± SE)
|
Hyper-hydricity
|
80 ml
|
50%
|
1.54 ± 0.04d
|
-52.74 ± 21.24c
|
NO (0%)
|
100 ml
|
100%
|
10.23 ± 0.24b
|
849.83 ± 76.21a
|
NO (0%)
|
150 ml
|
100%
|
12.92 ± 0.33a
|
894.31 ± 18.02a
|
NO (0%)
|
200 ml
|
100%
|
8.93 + 0.32c
|
632.49 ± 31.13b
|
YES (75%)
|
Mean values within a column with the same letter are not significantly different at P = 0.05 (Duncan’s Multiple Range Test) |
The shorter time of immersion (1 min and 2 min) turned out to be more favorable for the multiplication of shoots (8.92 ± 0.31 and 9.56 ± 0.22, respectively) and the production of twinflower biomass (721.15 ± 29.13 and 687.36 ± 36.32, respectively) (Table 4). Shoots showed no altered morphology.
Table 4
The influence of immersion frequency on L. borealis shoot biomass growth parameters using RITA® system of shoots immersed in a liquid medium
Immersion Frequency
|
Percentage
of Response
|
Multiplication
Rate
|
Fresh Biomass Growth Ratio (Mean ± SE)
|
Hyperhydricity
|
1 min. / 1h
|
100%
|
8.92 ± 0.31a
|
721.15 ± 29.13a
|
NO (0%)
|
2 min. / 1h
|
100%
|
9.56 ± 0.22a
|
687.36 ± 36.32a
|
NO (0%)
|
3 min. / 1h
|
100%
|
5.67 ± 0.13b
|
281.67 ± 17.07b
|
NO (0%)
|
Mean values within a column with the same letter are not significantly different at P = 0.05 (Duncan’s Multiple Range Test) |
For Linnaea borealis L., the growth of shoots in the RITA® bioreactor was highly efficient, especially when hormone supplementation in the medium was used (Table 2), the amount of medium in the culture vessel was 100 or 150 ml (Table 3), and the immersion time of the shoots in the medium was 1 or 2 min (Table 4).
Similar observations with RITA® system have been demonstrated by other authors. The highest shoot number of Stevia rebaudiana Bertoni, a medicinal plant containing steviol glycosides, was obtained from the RITA® bioreactor filled with 300 ml of medium in the culture vessel and the application of the immersion frequency of 10 s per 1 hour. In general, as a result of the gradual increase in the immersion frequency from 1 per 8 hours to 1 per 1 hour and the average medium volume from 100 ml to 300 ml in the culture vessel, the number of shoots per explant increased. When employing more volume of culture medium, vitrified shoots were formed, which showed abnormalities in their morphology and anatomy (Bayraktar 2019). Another species, Schisandra chinensis (Turcz.) Baill, a rich source of therapeutically important lignans with anticancer, immunostimulatory and hepatoprotective properties, showed the highest growth of shoot biomass in the RITA® system compared to four other breeding systems, including a balloon-type bioreactor or a Platform system. Growth of shoots in the RITA® bioreactor using 200 ml of medium and 5 min dipping periods once every 90 minutes resulted in good growth of healthy and correctly developed shoots, which gives hope for an easy expansion of the culture scale in the future (Szopa et al. 2017). In turn, in the RITA® bioreactor, immersion in 200 ml culture media lasting 15 minutes every 4 hours resulted in an approx. 1.8-fold increase in the biomass of the exotic species used in the production of perfumes and traditional medicine of Asian countries, Aquilaria malaccensis Lamk. The shoots were healthy and were not producing callus (Esyanti et al. 2019). Studies on in vitro shoot multiplication of Scutellaria alpine L., a species rich in polyphenol metabolites, show the benefits of using a temporary-immersion bioreactor. Experimentally selected parameters − 60 ml of the liquid medium in the culture vessel and the immersion time − 40 seconds influenced the quality and number of multiplied shoots. The results of the conducted research show a 1.5-fold increase in the number of cultured shoots and a 4-fold increase in the shoot biomass compared to the cultivation of the same species carried out on a solidified agar medium (Grzegorczak-Karolak et al. 2017).
The aim of this study was to determine the best system for propagating the twinflower shoots in the in vitro system - liquid media in an agitated system and in a temporary immersion bioreactor were used. Linnaea borealis L. produce lateral shoots of three kinds, vertical sexually reproductive shoots, non-sexually reproductive leafy shoots, and horizontal shoots (Niva et al. 2003). This species may develop efficiently new shoots in liquid media (in an agitated system and in a temporary immersion bioreactor), as the medium rinses the node fragments of all shoots, regardless of their location on the shoot. On a solid medium, there is no possibility of direct contact of media nutrients with nodes that do not straightway touch the medium (located on the higher parts of the stem), therefore they mainly multiply at the base node of the shoot. On the other hand, in liquid media, shoots also spread from higher placed nodes and horizontal shoots grow, which do not encounter any obstacles, and from them, new vertical shoots also grow. In horizontally spreading shoots, the nutrients may be resorbed by more parts simultaneously. Moreover, some observations argue that the population of this species during wet years clearly grows (Piękoś-Mirkowa and Mirek 2003). As it results from our observations of the wild population, this species prefers has been found in damp locations, within easy reach of running or dripping water (data not shown). For this reason, the system of shoots agitated or immersed in liquid media is an efficient system for the multiplication of the shoot biomass of this species, and is more preferred than a stationary system on solidified media.
One of the technologies of conservation of rare and endangered plants is short cold storage of encapsulated propagules. The formation of beads with appropriate stability and hardness is of key importance for producing somatic seeds: very hard beads limit the regeneration ability, while soft beads dissolve without protecting the encapsulated propagules. The type of explant, the concentrations of sodium alginate (SA) and calcium chloride (CC), and complexation duration were studied for L. borealis somatic seeds. Out of the two different concentrations of SA (3%, 4%) and the two concentrations of CC (100, 200 mM) evaluated to develop the encapsulation matrix, 3% and 4% SA and 200 mM CC were the most appropriate for beads production (Table 5). Therefore these two options (3% SA + 200 mM CC and 4% SA + 200 mM CC) were selected to be used in the experiment of short-term storage of propagules at lower (4°C) and low (-18 ºC) temperatures (Table 6).
Table 5
The influence of beat composition on synthetic seeds formation
SODIUM ALGINATE
|
CALCIUM CHLORIDE
|
BEAD CHARACTERISTIC
|
PURPOSE
|
3%
|
100 mM
|
Too soft to handle, formed tails
|
Not used for research
|
3%
|
200 mM
|
Isodiametric beads
|
Used for research
|
4%
|
100 mM
|
Isodiametric beads
|
Not used for research
|
4%
|
200 mM
|
Isodiametric beads
|
Used for research
|
Table 6
The influence of beat composition and storage duration (4°C, -18ºC) on the recovery of somatic seeds of L. borealis.
Sodium Alginate
|
Calcium Chloride
|
Storage Temperature
|
Storage
Duration
|
Survival
Percentage
|
Recovery Percentage ± SE
|
3%
|
200 mM
|
4°C
|
0 months
|
100%
|
100 ± 0.00%
|
3%
|
200 mM
|
4°C
|
2 months
|
80%
|
90 ± 3.7%
|
3%
|
200 mM
|
4°C
|
4 months
|
60%
|
73 ± 2.6%
|
3%
|
200 mM
|
4°C
|
6 months
|
60%
|
67 ± 2.6%
|
4%
|
200 mM
|
4°C
|
0 months
|
100%
|
98 ± 1.3%
|
4%
|
200 mM
|
4°C
|
2 months
|
100%
|
89 ± 2.7%
|
4%
|
200 mM
|
4°C
|
4 months
|
80%
|
78 ± 2.0%
|
4%
|
200 mM
|
4°C
|
6 months
|
70%
|
70 ± 2.6%
|
3%
|
200 mM
|
-18 ºC
|
0 months
|
100%
|
100 ± 0.0%
|
3%
|
200 mM
|
-18 ºC
|
2 months
|
80%
|
78 ± 2.0%
|
3%
|
200 mM
|
-18 ºC
|
4 months
|
60%
|
62 ± 2.5%
|
3%
|
200 mM
|
-18 ºC
|
6 months
|
60%
|
58 ± 2.9%
|
4%
|
200 mM
|
-18 ºC
|
0 months
|
100%
|
98 ± 1.3%
|
4%
|
200 mM
|
-18 ºC
|
2 months
|
100%
|
73 ± 1.5%
|
4%
|
200 mM
|
-18 ºC
|
4 months
|
80%
|
59 ± 2.3%
|
4%
|
200 mM
|
-18 ºC
|
6 months
|
70%
|
54 ± 2.2%
|
Mean values within a column with the same letter are not significantly different at P = 0.05 (Duncan’s Multiple Range Test) |
The survival and recovery rates, regardless of the encapsulation matrix used, subsequently decreased with the increased storage duration (from 100–60% and from 100–54%, respectively). Plants regenerated more efficiently when the explants, from which they developed, were stored at a higher temperature (4°C). The highest percentage of survival (100%) and recovery (98–100%) were obtained for beads inoculated immediately after formation (Table 6).
Somatic seeds production of a wide range of important endangered and protected species is considered an effective way to support their conservation. The maximum regeneration rate of 74% ± 2.72% was observed for axillary buds of Eryngium alpinum L., a protected alpine species, encapsulated in 4% sodium–alginate complexed with 300 mM calcium chloride after 2 months of storage at low temperature (Kikowska et al. 2020). Axillary buds of Rubus chamaemorus L., a glacial relict occurring in a few protected reserves, encapsulated in alginate hydrogel (5% SA and 50 µM CC) and stored at 4℃ for 3 months survived and regenerated in almost 56% (Thiem 2002). The storage potential of artificial seeds of Cymbidium aloifolium protocorms, a Threatened Orchid of Nepal, up to 90 days found 83.33% viability at 4°C storage on MS media (Pradhan et al. 2016). The results indicated that synseeds (3% SA, 100 mM CC) obtained from shoot tips of Taraxacum pieninicum Pawł. can be stored at 4°C even for 12 months (Kamińska et al. 2017).
Somatic seeds possess immense potential for large-scale production of plants as an alternative option to true seeds, have the potential to regenerate elite genotypes, and preserve important plant genetic resources (Nandini and Giridhar 2019). Artificial seed production and storage protocols allow the continuous supply of plant material of medicinal importance (Kikowska and Thiem 2011). Moreover, collections of in vitro cultures in combination with the methods of storing plant material provide tools that guarantee the protection of genetic resources of valuable plant species (Mikuła et al. 2013).