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Article

Effect of Liquid Culture Systems (Temporary Immersion Bioreactor and Rotary Shaker) Used During Multiplication and Differentiation on Efficiency of Repetitive Somatic Embryogenesis of Narcissus L. ‘Carlton’

by
Małgorzata Malik
,
Ewelina Tomiak
* and
Bożena Pawłowska
Department of Ornamental Plants and Garden Art, University of Agriculture in Krakow, 29-Listopada 54, 31-425 Kraków, Poland
*
Author to whom correspondence should be addressed.
Agronomy 2025, 15(1), 85; https://doi.org/10.3390/agronomy15010085
Submission received: 13 November 2024 / Revised: 23 December 2024 / Accepted: 30 December 2024 / Published: 31 December 2024
(This article belongs to the Special Issue Plant Tissue Culture and Plant Somatic Embryogenesis)
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Abstract

:
Liquid culture systems, including bioreactors, are valuable tools for the scaling up of production. Their involvement leads to the automation of the highly efficient, reproducible somatic embryogenesis of Narcissus L. ‘Carlton’. Alternative procedures for efficient embryogenic tissue and early somatic embryo multiplication have been developed. The long-term embryogenic callus of narcissus ‘Carlton’, obtained by repetitive somatic embryogenesis, was multiplicated and differentiated in different liquid culture systems. For multiplication, the Rita® temporary immersion bioreactor and the rotary shaker at 60 rpm and 100 rpm were used, and, for differentiation, the rotary shaker at 60 rpm and solid cultures were investigated. Cultures immersed with a frequency of 15 min every 24 h during multiplication were characterized by the greatest increase in biomass (1.3), and the greatest number of embryos (152.6 embryos per 1 g of inoculum) was seen during differentiation. Higher immersion frequencies (15 min every 8 and 12 h) decreased the tissue quality and yield. The use of a bioreactor during multiplication promoted the number of embryos obtained during differentiation. In turn, cultivation in a rotary shaker during differentiation, regardless of the multiplication system, stimulated the multiplication of embryogenic tissue. The liquid medium used for the multiplication and differentiation of somatic embryos improved the synchronization of their development, which reached up to 95–99% depending on the system.

1. Introduction

Narcissus L. is a very important long-lived perennial geophyte from a botanical, horticultural, folkloric, and medicinal perspective [1,2,3]. These bulbous plants are widely used as ornamental plants in urban landscaping and home gardens and as a cut flower. Bulbs are produced on a large scale throughout Europe, especially in the Netherlands, and globally [4,5].
In recent years, daffodils have gained enormous popularity due to the content of biologically active compounds in their bulbs, and they are a rich source of specialized metabolites. Alkaloids such as galanthamine (Gal), lycorine, and narciclasine are known for their pharmaceutical properties; they are used during therapy in Alzheimer’s [6] and cancer [7]. Narcissus pseudonarcissus L. ‘Carlton’ was chosen as a source due to the relatively high concentration of Gal in the bulbs [1,3,4,8,9]. Plant tissue culture is a promising alternative to optimize alkaloid extraction [6,7]. Ferdausi’s [10,11] studies also confirmed the presence of galantamine in bulbs, shoots, and calli obtained in vitro from the ‘Carlton’ cultivar at high auxin concentrations.
In vitro cultures are used for the mass production of plants that are difficult to propagate traditionally. They provide an opportunity to obtain elite propagation material and are also helpful in the propagation of plants with industrial and pharmacological potential, as well as the propagation of endangered species and their cryopreservation [12,13,14,15,16]. Under field conditions, the natural vegetative propagation rate of narcissus when separating adventitious bulbs is very slow, at nearly 1.6 bulbs/year [17,18]. Chipping and twin-scaling are two traditional propagation techniques for rapid reproduction, which are more efficient but are insufficient and do not meet the current needs of mass production [19,20].
In vitro propagation is a technique that includes somatic embryogenesis, which is one of the basic tools that is widely used in the high-yield reproduction of many species. In the genus Narcissus, somatic embryogenesis has been achieved in Narcissus confusus [21]; Narcissus pseudonarcissus cv. ‘Golden Harvest’, ‘St. Keverne’ [22], and ‘Carlton’ [23]; Narcissus tazzeta [24,25]; and Narcissus papyraceus cv. Shirazi [26].
Repetitive somatic embryogenesis (RSE) offers even greater opportunities for the automation and commercialization of production due to the scale-up of production through the continuous multiplication of somatic embryos [27,28]. New embryos can proliferate continuously through consecutive cycles of secondary somatic embryogenesis. Embryos are formed from embryos that were previously formed [27,29]. The advantages of long-term embryogenic cultures obtained in this way are adaptability to different liquid culture systems, efficient handling, and the independence of this process from the original explant source [30]. High-frequency secondary and repetitive somatic embryogenesis has been developed for Coffea arabica [31], Camellia assamica [32], Quercus robur [33], Camellia sinensis [30], and Hepatica nobilis [34]. High-yielding RSE for narcissus cv. Carlton for solid cultures has also been developed [29].
The use of liquid culture systems for propagation by somatic embryogenesis provides many benefits, including increased efficiency, increased nutrient uptake, more uniform culture conditions, more homogeneous material, and reduced production costs [31,35]. A special system consists of a bioreactor designed for intensive plant propagation in liquid media under controlled environmental conditions. In the Rita® temporary immersion bioreactor, these include the time of tissue contact with the media, aeration, and air exchange [36]. The method of operation of temporary immersion systems involves periodically immersing the cultured tissue in a liquid medium, followed by draining and exposing the plant tissue to a sterile gaseous environment [35]. Temporary immersion and gas exchange in the bioreactor vessel allow one to overcome the occurrence of hyperhydricity or asphyxia, which is often observed in cultures continuously immersed in a liquid medium [37].
To date, for narcissus ‘Carlton’, a protocol involving liquid media for the initiation of somatic embryogenesis has been developed. Somatic embryogenesis was induced in ovary cultures by alternating the use of liquid media and solid media [38]. The aim of the present study was to check whether the use of different liquid systems (rotary shaker and TIS technology) during multiplication and differentiation would increase the efficiency of narcissus RSE, the synchronization of somatic embryos, and biomass production.

2. Materials and Methods

2.1. Plant Material

A long-term (seven-year-old) embryogenic callus obtained by RSE from ovary explants isolated from Narcissus L. ‘Carlton’ flower buds originating from bulbs (12 cm in circumference) chilled for 3 weeks at 5 °C was taken for the experiments [29]. The callus was initiated and grown on solid Murashige and Skoog (MS) medium [39] with 25 µM Picloram and 5 µM 6-benzylaminopurine (BA) and 3% sucrose. The medium was adjusted to pH 5.5 before autoclaving and was gelled with 0.5% agar (Lab-Agar™, Biocorp, Warsaw, Poland). The callus was maintained at 20 ± 2 °C in darkness. Cultures were transferred to fresh medium every 4–6 weeks.

2.2. Effect of Immersion Frequency—Experiment 1

A two-stage experiment was assumed: six-week multiplication followed by six-week differentiation stages. For multiplication, clusters of embryogenic tissue (callus with differentiating primary and secondary embryos at the globular stage) were placed in the vessels of a Rita® bioreactor (CIRAD, Montpellier, France) with a temporary immersion system filled with 200 mL of multiplication liquid medium. Experiment 1 was designed as a single-factor experiment (immersion frequency used during multiplication) and investigated the influence of the factor on the RSE performance during the multiplication and differentiation stages. The effect of the following immersion frequencies was investigated: (i) 15 min every 24 h (TIS 1 × 15), (ii) 15 min every 8 h (TIS 3 × 15), and (iii) 15 min every 2 h (TIS 12 × 15). The control involved cultures in Petri dishes (diameter 9 cm, height 2.5 cm) filled with 25 mL of multiplication agar-solidified medium (0.5%, Lab-Agar™, Biocorp, Warsaw, Poland). The multiplication medium contained macro- and micronutrients and vitamins, as described by Murashige and Skoog [39], as well as 25 µM Picloram, 5 µM BA, and 3% sucrose. The medium was adjusted to pH 5.5 before autoclaving. Each Petri dish was filled with 2 g of embryogenic tissue and each Rita® vessel was filled with 5 g. Cultures were maintained at 20 ± 2 °C in darkness.
For the differentiation stage, embryogenic tissue (2 g per dish) obtained in all combinations of the multiplication stage was transferred to a Petri dish (diameter 9 cm, height 2.5 cm) on solid MS medium with 5 µM BA and 0.5 µM naphthalene-1-acetic acid (NAA) and 3% sucrose. The medium was adjusted to pH 5.8 before autoclaving and was gelled with 0.5% agar (Lab-Agar™, Biocorp, Warsaw, Poland). The callus was maintained at 20 ± 2 °C in darkness.

2.3. Effect of Liquid Culture System—Experiment 2

Experiment 2 was designed as a two-factor experiment (culture system used during multiplication × culture system used during differentiation) and investigated the influence of both factors on the RSE performance during the differentiation stage. Embryogenic tissue multiplied for six weeks in three liquid systems was used for experiment 2: (i) continuous cultivation in liquid medium on a rotary shaker at 60 rpm (RS 60), (ii) continuous cultivation in liquid medium on a rotary shaker at 100 rpm (RS 100), (iii) temporary immersion system at an immersion frequency of 15 min every 24 h (TIS 1 × 15).
Clusters of embryogenic callus obtained during multiplication (on multiplication medium) for regeneration were transferred to MS regeneration medium containing 5 µM BA and 0.5 µM NAA and 3% sucrose. Calli were cultured for six weeks in two ways: continuously in liquid medium on a rotary shaker at 60 rpm or on solid medium and then for six weeks on solid medium of the same composition. Solid media were gelled with 0.5% agar (Lab-Agar™, Biocorp, Warsaw, Poland). Control cultures were maintained on solid media during multiplication and differentiation.
The following cultivation vessels were used: 100 mL Erlenmeyer flasks with 20 mL of medium and 1 g of embryogenic tissue for cultivation on a rotary shaker and Petri dishes (diameter 9 cm, height 2.5 cm) with 20 mL of solid medium and 2 g of tissue for cultivation on solid medium. Cultures were maintained at 20 ± 2 °C in darkness.

2.4. Growth Evaluation and Statistical Analysis

After six and 12 weeks, the biomass growth index and the total number of somatic embryos per 1 g of inoculum tissue were calculated. For the biomass growth index, the following formula was used: (FFW − IFW)/IFW, where FFW = final fresh weight and IFW = initial fresh weight. The percentage shares of the individual developmental stages (early, mature and cotyledonary) in the total number of embryos obtained were also determined. The percentage of early embryos in the total number of embryos was defined as synchronization.
The results of the observations were evaluated by analysis of variance. Means that differed significantly were identified using Tukey’s multiple test at a significance level of p ≤ 0.05 (Statistica version 10, StatSoft, Kraków, Poland).

3. Results and Discussion

3.1. Biomass Growth

The scale-up of production resulting from the involvement of RSE can be achieved due to faster tissue growth and easy adaptation to liquid culture systems [32,40,41]. The direct contact of plant cells with the liquid medium, i.e., the better availability of nutrients and growth regulators, allows for increased efficiency [42] but also influences the synchronization of production [31].
In the narcissus ‘Carlton’ cultures, the multiplication of the embryogenic tissue and the differentiation of somatic embryos were observed in all culture systems tested. The decisive parameters were the state of the medium and the time of contact with the liquid medium. The long-term callus representing RSE, continuously multiplied on solid medium containing 25 µM Picloram and 5 µM BA after transfer to the Rita® bioreactor in liquid multiplication medium, proliferated best when the frequency of immersion was 15 min every 24 h (TIS 1 × 15). The multiplication rate was 1.3, which was higher compared to that obtained on solid media, i.e., TIS 3 × 15 and TIS 12 × 15 (Table 1 and Figure 1a). However, the lowest biomass increase was observed in cultures immersed for 15 min every 8 h (0.2, TIS 3 × 15). In the cultures that were immersed the most frequently, the dedifferentiation of the embryogenic tissue was observed more often, as well as its faster ageing (Figure 1b).
When transferring the callus cultures from the bioreactor to a solid regeneration medium with 5 µM BA and 0.5 µM NAA, the biomass growth in the TIS 3 × 15 cultures (0.7) was still the lowest, while intensified growth was observed in the control on solid medium (2.1). This resulted not only from the proliferation of the embryogenic tissue but also from the development of somatic embryos and their conversion (Figure 1c).
The efficiency of embryogenic callus proliferation and somatic embryo differentiation in the Rita® temporary immersion bioreactor has been repeatedly reported to be highly dependent on the immersion frequency. As a rule, more frequent immersions promoted an increase in fresh weight, which was not observed in the case of the embryogenic callus of narcissus. In cork oak [43], an immersion frequency of 1 min every 6 or 4 h increased the fresh weight compared to a frequency of 1 min every 12 h and with semi-solid media. Frequencies of 1 min every 6, 8 and 12 h increased the biomass growth of Quercus robur embryo clusters compared to a solid medium [33]. The immersion frequency of 5 min every 4 h, compared to 1, 10 and 15 min every 4 h and with a semi-solid medium, promoted the multiplication of secondary somatic embryos of Eurycoma longifolia. Increased productivity was also observed when the immersion frequency was changed from every 4 h to every 8 h [44].
An increase in biomass when using liquid systems can also be achieved at the differentiation stage. After a differentiation cycle in various culture systems, i.e., solid and rotary shakers at 60 rpm, significant differences in narcissus biomass growth were observed. Regardless of the culture system used during the multiplication of the narcissus embryogenic tissue (RS 60, RS 100, TIS 1 × 15, solid medium—control), a higher or similar biomass growth index was obtained in shaking cultures compared to solid cultures (Table 2).
The shaking speed (60 or 100 rpm) used during multiplication only influenced biomass growth during the differentiation stage. After 12 weeks of differentiation, significantly more embryogenic calli were observed in cultures shaken at 60 rpm compared to 100 rpm (Table 2).
The highest biomass growth was noted in cultures grown on solid media, which were transferred to liquid media and shaken at 60 rpm (solid RS60) after both 6 and 12 weeks of cultivation (5.9-fold and 11.7-fold increase, respectively). The solid differentiation medium used for this material did not favor the multiplication of the embryogenic tissue. Biomass growth on the solid medium amounted to 2.2 and 4.7, respectively. Transferring the tissue grown in liquid media (systems: RS60, RS100, TIS 1 × 15) to solid media for differentiation inhibited multiplication. On the other hand, continuation in liquid media supported the multiplication of embryogenic tissue.

3.2. Somatic Embryo Differentiation

The differentiation of somatic embryos occurs when the auxin concentration in the medium decreases [38,45]. Reducing the auxin concentration allows the achievement of subsequent embryo developmental stages: globular, torpedo, mature, cotyledonary and converted plants [35]. In the ‘Carlton’ cultures maintained on a multiplication medium in a bioreactor, the largest number of embryos was formed when the embryogenic tissue had the least frequent contact with a medium with high auxin content, i.e., for an immersion frequency of 1 × 15 min or 3 × 15 min per day (51.7 embryos and 61.9 embryos, respectively; Table 1). In combinations where contact with the multiplication medium was longer, in solid and bioreactor cultures immersed for 12 × 15 min per day, the smallest number of embryos was formed (49.2 and 41.8, respectively). Further differentiation of the somatic embryos was observed on a solid regeneration medium with the reduced availability of auxin. Regarding the differentiation medium, the largest number of embryos was obtained in the cultures immersed the least frequently at the multiplication stage (1 × 15 min during the day)—152.6 embryos (Table 1).
Based on the results of experiment 2, it was found that the number of somatic embryos formed during differentiation depended on the culture system used at the multiplication stage (Table 2). Regardless of the other factors, the largest number of embryos was obtained in cultures multiplied in the Rita® bioreactor. The efficiency of embryo formation is also influenced by the culture system used immediately after multiplication. More embryos are observed when differentiation takes place on a solid medium. A similar relationship has been observed in the development of somatic embryos in many plant species, including Arabica coffee [31], peanut [45] and hybrid larch [46]. In the liquid medium (RS60) at the differentiation stage, the number of embryos was lower than that obtained on the solid medium in the case of cultures multiplied in the bioreactor (Table 2).

3.3. Somatic Embryo Maturation

The greatest diversity in the developmental stages among the narcissus embryos occurred in cultures grown on solid media, where all developmental stages of embryos appeared: globular, torpedo, mature, cotyledons and post-conversion (Figure 1d–f and Figure 2).
On solid media, in contrast to liquid media, where the cultures are more homogeneous, a concentration gradient of growth regulators is created, which causes somatic embryos to develop asynchronously [47]. This is due to the gradual consumption of auxin from the medium and the formation of a concentration gradient of growth regulators in clusters of embryogenic tissue with RSE. The decrease in the stimulation of endogenous IAA synthesis induced by high concentrations of exogenous auxin releases the potential for embryo formation and maturation [48]. In turn, maintaining a high concentration of auxin is necessary to maintain the embryogenic nature of embryogenic cultures of monocotyledonous plants [49]. When the somatic embryos were continuously or frequently immersed in the liquid medium, embryo maturation was delayed. Somatic embryos were arrested in the globular stage. This phenomenon allows for the long-term (from several months to several years) multiplication of embryogenic tissue in the process of RSE and is beneficial at the stage of the highly efficient proliferation of embryogenic tissue.
During the multiplication of narcissus in the Rita® bioreactor, synchronization of 90–99% (early stages of embryos) was observed. Similar values were achieved after transferring the material from the bioreactor to the solid regeneration medium (94–98% of embryos in early stages; Figure 1g). In the control cultures on solid media in the multiplication and differentiation stages, further embryo stages appeared. Synchronization was 72 and 74%, respectively (Figure 2).
Experiment 2 also confirmed that synchronization depended on the culture system used in the multiplication or differentiation stage. The narcissus ‘Carlton’ cultures that were propagated in the liquid culture systems (RS 60, RS 100 or TIS 1 × 15) after transfer to the regeneration medium, regardless of whether it was solid or liquid, maintained a high degree of synchronization for 6 weeks (77–95% of early-stage embryos). Further cultivation on solid regeneration medium led to the maturation of the embryos. The cotyledonary stage was observed in all combinations (1–20%), and the synchrony ranged from 37% in the control to 68–76% in cultures grown in the bioreactor (TIS 1 × 15) (Figure 3). Transferring the cultures to light for another 8 weeks stimulated further embryo development and conversion to plants (Figure 1h).
The commercial application of somatic embryogenesis has many limitations. One of the factors that makes it difficult to automate plant reproduction through somatic embryogenesis is the asynchronous development of embryos. Somatic embryogenesis consists of five stages: the initiation of embryogenesis, the proliferation of embryogenic cultures, differentiation—somatic embryo formation and early development, the maturation of somatic embryos and the conversion of embryos into plantlets [35,50]. These require the development of optimal factors (physical, chemical and biological) that enable the somatic embryos to achieve subsequent developmental stages synchronously at the same time. At the stage of embryo formation and its early development, in addition to influencing the composition of growth regulators in the medium, especially auxins, we can significantly influence the processes by selecting a liquid culture system. Temporary immersion systems that allow for the semi-automation or full automation of multi-stage regeneration processes are of particular importance. The use of the bioreactor for the RSE tissue of narcissus ‘Carlton’ allowed for the long-term multiplication of the embryogenic callus and early somatic embryos, effectively inhibiting embryo maturation above the globular stage. The goal for the future will be to develop a system or set of factors for the synchronous maturation and conversion of multiplied somatic embryos.

4. Conclusions

Based on the presented results, it can be stated that the highest efficiency of the RSE of narcissus ‘Carlton’ was obtained using the Rita bioreactor at the multiplication stage. After 6 weeks of immersion with a frequency of 15 min every 24 h, with a medium containing 25 µM Picloram and 5 µM BA, the biomass growth was 1.3. The highest efficiency of embryo differentiation (152.6 embryos per 1 g of inoculum) and, at the same time, the highest synchronization (98%) can be achieved by transferring the tissue multiplied in the bioreactor to a solid medium containing 5 µM BA and 0.5 µM NAA for the next 6 weeks. Carrying out differentiation in rotary shaker cultures stimulates multiplication while reducing the number of somatic embryos obtained.

Author Contributions

Conceptualization, M.M.; methodology, M.M. and E.T.; software, M.M.; validation, M.M. and B.P.; formal analysis, M.M. and E.T.; investigation, M.M. and E.T.; resources, M.M.; data curation, M.M. and E.T.; writing—original draft preparation, M.M. and E.T.; writing—review and editing, M.M. and E.T.; visualization, M.M. and E.T.; supervision, M.M.; funding acquisition, B.P.; project administration, M.M., B.P. and E.T. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the Ministry of Science and Higher Education of the Republic of Poland from subvention funds for the University of Agriculture in Krakow.

Data Availability Statement

The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Křoustková, J.; Ritomská, A.; Al Mamun, A.; Hulcová, D.; Opletal, L.; Kuneš, J.; Bucar, F. Structural analysis of unusual alkaloids isolated from Narcissus pseudonarcissus cv. ‘Carlton’. Phytochemistry 2022, 204, 113439. [Google Scholar] [CrossRef] [PubMed]
  2. Jezdinský, A.; Jezdinská Slezák, A.; Vachůn, A.; Pokluda, R.; Uher, J. Effect of saline water on the vase life of Narcissus poeticus L. flowers. Folia Hortic. 2024, 36, 95–117. [Google Scholar] [CrossRef]
  3. Boshra, Y.R.; Fahim, J.R.; Hamed, A.N.E.; Desoukey, S.Y. Phytochemical and biological attributes of Narcissus pseudonarcissus L. (Amaryllidaceae): A review. S. Afr. J. Bot. 2022, 146, 437–458. [Google Scholar] [CrossRef]
  4. Berkov, S.; Georgieva, L.; Sidjimova, B.; Nikolova, M.; Stanilova, M.; Bastida, J. In vitro propagation and biosynthesis of Sceletium-type alkaloids in Narcissus pallidulus and Narcissus cv. ‘Hawera’. S. Afr. J. Bot. 2021, 136, 190–194. [Google Scholar] [CrossRef]
  5. The Daffodil Society. Bulb Suppliers. 2019. Available online: https://thedaffodilsociety.com/bulb-suppliers/ (accessed on 5 December 2019).
  6. Santos, G.S.; Sinoti, S.B.P.; de Almeida, F.T.C.; Silveira, D.; Simeoni, L.A.; Gomes-Copeland, K.K.P. Use of galantamine in the treatment of Alzheimer’s disease and strategies to optimize its biosynthesis using the in vitro culture technique. Plant Cell Tissue Organ Cult. (PCTOC) 2020, 143, 13–29. [Google Scholar] [CrossRef]
  7. Sena, S.; Kaur, H.; Kumar, V. Lycorine as a lead molecule in the treatment of cancer and strategies for its biosynthesis using the in vitro culture technique. Phytochem. Rev. 2024, 23, 1861–1888. [Google Scholar] [CrossRef]
  8. Lubbe, A.; Gude, H.; Verpoorte, R.; Choi, Y.H. Seasonal accumulation of major alkaloids in organs of pharmaceutical crop Narcissus ‘Carlton’. Phytochemistry 2013, 88, 43–53. [Google Scholar] [CrossRef]
  9. Georgiev, V.; Ivanov, I.; Pavlov, A. Recent Progress in Amaryllidaceae Biotechnology. Molecules 2020, 25, 4670. [Google Scholar] [CrossRef] [PubMed]
  10. Ferdausi, A.; Chang, X.; Hall, A.; Jones, M. Galanthamine production in tissue culture and metabolomic study on Amaryllidaceae alkaloids in Narcissus pseudonarcissus cv. ‘Carlton’. Ind. Crops Prod. 2020, 144, 112058. [Google Scholar] [CrossRef]
  11. Ferdausi, A.; Chang, X.; Jones, M. Enhancement of galanthamine production through elicitation and NMR-based metaboliteprofiling in Narcissus pseudonarcissus cv. Carlton in vitro callus cultures. Vitr. Cell. Dev. Biol. Plant 2021, 57, 435–446. [Google Scholar] [CrossRef]
  12. Juan Vicedo, J.; Pavlov, A.; Ríos, S.; Casas, J.L. Micropropagation of five endemic, rare and/or endangered Narcissus species from the Iberian Peninsula (Spain and Portugal). Acta Biol. Crac. Ser. Bot. 2021, 63, 55–61. [Google Scholar] [CrossRef]
  13. Abu Zahra, H.M.F.; Oran, S.A. Micropropagation of the wild endangered daffodil Narcissus tazetta L. Acta Hortic. 2009, 826, 135–140. [Google Scholar] [CrossRef]
  14. Sage, D.O. Propagation and protection of flower bulbs: Current approaches and future prospects, with special reference to Narcissus. Acta Hortic. 2005, 673, 323–334. [Google Scholar] [CrossRef]
  15. Kocot, D.; Nowak, B.; Sitek, E. Long-term organogenic callus cultivation of Ranunculus illyricus L.: A blueprint for sustainable ex situ conservation of the species in urban greenery. BMC Plant Biol. 2024, 24, 212. [Google Scholar] [CrossRef] [PubMed]
  16. Maślanka, M.; Panis, B.; Malik, M. Cryopreservation of Narcissus L. ‘Carlton’ somaticembryos by dropletvitrification. Propag. Ornam. Plants 2016, 16, 28–35. [Google Scholar]
  17. Rees, A.R. The initiation and growth of Narcissus bulbs. Ann. Bot. 1969, 33, 277–288. [Google Scholar] [CrossRef]
  18. Slezák, K.A.; Mazur, J.; Jezdinský, A.; Kapczyńska, A. Bulb size interacts with lifting term in determining the quality of Narcissus poeticus L. propagation material. Agronomy 2020, 10, 975. [Google Scholar] [CrossRef]
  19. Rees, A.R. Ornamental Bulbs, Corms and Tubers; CABI International: Wallingford, UK, 1992; p. 220. [Google Scholar]
  20. Hanks, G.R. Narcissus and Daffodil: The Genus Narcissus; CRC Press: London, UK, 2002; p. 452. [Google Scholar] [CrossRef]
  21. Selles, M.; Viladomat, F.; Bastida, J.; Codina, C. Callus induction, somatic embryogenesis and organogenesis in Narcissus confusus: Correlation between the state of differentiation and the content of galanthamine and related alkaloids. Plant Cell Rep. 1999, 18, 646–651. [Google Scholar] [CrossRef]
  22. Sage, D.O.; Lynn, J.; Hammatt, N. Somatic embryogenesis in Narcissus pseudonarcissus cvs. Golden Harvest and St. Keverne. Plant Sci. 2000, 150, 209–216. [Google Scholar] [CrossRef]
  23. Malik, M.; Bach, A. Morphogenetic pathways from Narcissus L. ‘Carlton’ in vitro cultures of pcstage flower bud explants according to cytokinin and auxin ratios. Acta Sci. Pol. Hortorum Cultus 2016, 15, 101–111. [Google Scholar]
  24. Chen, L.; Zhu, X.; Gu, L.; Wu, J. Efficient callus induction and plant regeneration from anther of Chinese narcissus (Narcissustazetta L. var. chinensis Roem). Plant Cell Rep. 2005, 24, 401–407. [Google Scholar] [CrossRef]
  25. Al-Zghoul, T.M.; Shibli, R.A.; Qudah, T.S.; Tahtamouni, R.W.; Rawshdeh, N. Employment of Somatic Embryogenesis as a Tool for Rescuing Imperiled Narcissus tazetta L. Growing Wild in Jordanian Environment. Jordan J. Biol. Sci. 2021, 14, 559–564. [Google Scholar] [CrossRef]
  26. Anbari, S.; Tohidfar, M.; Hosseini, R.; Haddad, R. Somatic embryogenesis induction in Narcissus papyraceus cv. Shirazi. Plant Tissue Cult. Biotechnol. 2007, 17, 37–46. [Google Scholar] [CrossRef]
  27. Raemakers, C.J.J.M.; Jacobsen, E.A.; Visser, R.G.F. Secondary somatic embryogenesis and applications in plant breeding. Euphytica 1995, 81, 93–107. [Google Scholar] [CrossRef]
  28. Vasic, D.; Alibert, G.; Skoric, D. Protocols for efficient repetitive and secondary somatic embryogenesis in Helianthus maximiliani (Schrader). Plant Cell Rep. 2001, 20, 121–125. [Google Scholar] [CrossRef]
  29. Malik, M.; Bach, A. High-yielding repetitive somatic embryogenesis in cultures of Narcissus L. ‘Carlton’. Acta Sci. Pol. Hortorum Cultus 2017, 16, 107–112. [Google Scholar]
  30. Akula, A.; Becker, D.; Bedson, M. High-yielding repetitive somatic embryogenesis and plant recovery in a selected tea clone, TRI-2025, by temporary immersion. Plant Cell Rep. 2001, 19, 1140–1145. [Google Scholar] [CrossRef] [PubMed]
  31. Aguilar, M.E.; Wang, X.-Y.; Escalona, M.; Yan, L.; Huang, L.-F. Somatic embryogenesis of Arabica coffee in temporary immersion culture: Advances, limitations, and perspectives for mass propagation of selected genotypes. Front. Plant Sci. 2022, 13, 994578. [Google Scholar] [CrossRef]
  32. Bajpai, R.; Chaturvedi, R. Recurrent secondary embryogenesis in androgenic embryos and clonal fidelity assessment of haploid plants of Tea, Camellia assamica ssp. assamica and Camellia assamica ssp. lasiocaylx. Plant Cell Tissue Organ Cult. 2021, 145, 127–141. [Google Scholar] [CrossRef]
  33. Mallón, R.; Covelo, P.; Vieitez, A.M. Improving secondary embryogenesis in Quercus robur: Application of temporary immersion for mass propagation. Trees 2012, 26, 731–741. [Google Scholar] [CrossRef]
  34. Szewczyk-Taranek, B.; Pawłowska, B. Recurrent somatic embryogenesis and plant regeneration from seedlings of Hepatica nobilis Schreb. Plant Cell Tissue Organ Cult. 2015, 120, 1203–1207. [Google Scholar] [CrossRef]
  35. Murthy, H.N.; Joseph, K.S.; Paek, K.Y.; Park, S.Y. Bioreactor systems for micropropagation of plants: Presentscenario and futureprospects. Front. Plant Sci. 2023, 14, 1159588. [Google Scholar] [CrossRef] [PubMed]
  36. Berthouly, M.; Etienne, H. Temporary immersion system: A new concept for use liquid medium in mass propagation. In Liquid Culture Systems for In Vitro Plant Propagation; Springer: Dordrecht, The Netherlands, 2005; pp. 165–195. [Google Scholar] [CrossRef]
  37. Etienne, H.; Berthouly, M. Temporary immersion systems in plant micropropagation. Plant Cell Tissue Organ Cult. 2002, 69, 215–231. [Google Scholar] [CrossRef]
  38. Malik, M. Comparison of different liquid/solid culture systems in the production of somatic embryos from Narcissus L. ovary explants. Plant Cell Tissue Organ Cult. 2008, 94, 337–345. [Google Scholar] [CrossRef]
  39. Murashige, T.; Skoog, F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 1962, 15, 473–497. [Google Scholar] [CrossRef]
  40. Burns, J.A.; Wetzstein, H.Y. Development and characterization of embryogenic suspension cultures of pecan. Plant Cell Tissue Organ Cult. 1997, 48, 93–102. [Google Scholar] [CrossRef]
  41. Guan, Y.; Li, S.-G.; Fan, X.-F.; Su, Z.-H. Application of Somatic Embryogenesis in Woody Plants. Front. Plant Sci. 2016, 7, 938. [Google Scholar] [CrossRef] [PubMed]
  42. Ziv, M. Simple bioreactors for mass propagation of plants. In Liquid Culture Systems for In Vitro Plant Propagation; Hvoslef-Eide, A.K., Preil, W., Eds.; Springer: Dordrecht, The Netherlands, 2005. [Google Scholar] [CrossRef]
  43. Pérez, M.; Bueno, M.A.; Escalona, M.; Toorop, P.; Rodríguez, R.; Cañal, M.J. Temporary immersion systems (RITA®®) for the improvement of cork oak somatic embryogenic culture proliferation and somatic embryo production. Trees 2013, 27, 1277–1284. [Google Scholar] [CrossRef]
  44. Mohd, N.M.; Ja’Afar, H.; Zawawi, D.D.; Alias, N. In Vitro Somatic Embryos Multiplication of Eurycoma longifolia Jack using Temporary Immersion System RITA®®. Sains Malays. 2017, 46, 897–902. [Google Scholar] [CrossRef]
  45. Durham, R.E.; Parrott, W.A. Repetitive somatic embryogenesis from peanut cultures in liquid medium. Plant Cell Rep. 1992, 11, 122–125. [Google Scholar] [CrossRef] [PubMed]
  46. Le, K.C.; Dedicova, B.; Johansson, S.; Lelu-Walter, M.A.; Egertsdotter, U. Temporary immersion bioreactor system for propagation by somatic embryogenesis of hybrid larch (Larix × eurolepis Henry). Biotechnol. Rep. 2021, 32, 684. [Google Scholar] [CrossRef] [PubMed]
  47. Li, F.; Yao, J.; Hu, L.; Chen, J.; Shi, J. Multiple Methods Synergistically Promote the Synchronization of Somatic Embryogenesis Through Suspension Culture in the New Hybrid Between Pinus elliottii and Pinus caribaea. Front. Plant Sci. 2022, 13, 857972. [Google Scholar] [CrossRef] [PubMed]
  48. Michalczuk, L.; Ribnicky, D.M.; Cooke, T.J.; Cohen, J.D. Regulation of indole-3-acetic acid biosynthetic pathways in carrot cell cultures. Plant Physiol. 1992, 100, 1346–1353. [Google Scholar] [CrossRef] [PubMed]
  49. Krishna, R.S.; Vasil, I.K. Somatic embryogenesis in herbaceous monocots. In In Vitro Embryogenesis in Plants, 1st ed.; Thorpe, T.A., Ed.; Kluwer Academic Publishers: Dordrecht, The Netherlands, 1995; pp. 471–540. [Google Scholar] [CrossRef]
  50. von Arnold, S.; Sabala, I.; Bozhkov, P.; Dyachok, J.; Filonova, L. Developmental pathways of somatic embryogenesis. Plant Cell Tissue Organ Cult. 2002, 69, 233–249. [Google Scholar] [CrossRef]
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Figure 1. Repetitive somatic embryogenesis of Narcissus L. ‘Carlton’ in different liquid culture systems. (a) Embryogenic tissue during multiplication in Rita® bioreactor immersed with frequency of 15 min every 24 h. Bar = 1 cm. (b) Embryogenic tissue during multiplication in Rita® bioreactor immersed with frequency of 15 min every 2 h. Bar = 1 cm. (c) Differentiation on solid medium (control cultures). (d) Early stages (globular and torpedo) of somatic embryos and bioreactor cultures (TIS 1 × 15) after transfer from rotary shaker at 60 rpm to solid medium. (e) Mature somatic embryo. (f) Cluster of embryogenic tissue with cotyledonary embryos. (g) Bioreactor cultures (TIS 1 × 15) after transfer to solid medium. Bar = 1 cm. (h) Embryo conversion in light—20th week of culture. Bar = 1 cm.
Figure 1. Repetitive somatic embryogenesis of Narcissus L. ‘Carlton’ in different liquid culture systems. (a) Embryogenic tissue during multiplication in Rita® bioreactor immersed with frequency of 15 min every 24 h. Bar = 1 cm. (b) Embryogenic tissue during multiplication in Rita® bioreactor immersed with frequency of 15 min every 2 h. Bar = 1 cm. (c) Differentiation on solid medium (control cultures). (d) Early stages (globular and torpedo) of somatic embryos and bioreactor cultures (TIS 1 × 15) after transfer from rotary shaker at 60 rpm to solid medium. (e) Mature somatic embryo. (f) Cluster of embryogenic tissue with cotyledonary embryos. (g) Bioreactor cultures (TIS 1 × 15) after transfer to solid medium. Bar = 1 cm. (h) Embryo conversion in light—20th week of culture. Bar = 1 cm.
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Figure 2. The effect of the immersion frequency on the percentage share of individual developmental stages of Narcissus L. ‘Carlton’ embryos (early, mature and cotyledonary) in the total number of embryos obtained during multiplication and differentiation (Solid—solid medium (control); TIS 1 × 15—cultures in Rita® bioreactor with immersion frequency of 15 min every 24 h; TIS 3 × 15—cultures in Rita® bioreactor with immersion frequency of 15 min every 8 h; TIS 12 × 15—cultures in Rita® bioreactor with immersion frequency of 15 min every 2 h).
Figure 2. The effect of the immersion frequency on the percentage share of individual developmental stages of Narcissus L. ‘Carlton’ embryos (early, mature and cotyledonary) in the total number of embryos obtained during multiplication and differentiation (Solid—solid medium (control); TIS 1 × 15—cultures in Rita® bioreactor with immersion frequency of 15 min every 24 h; TIS 3 × 15—cultures in Rita® bioreactor with immersion frequency of 15 min every 8 h; TIS 12 × 15—cultures in Rita® bioreactor with immersion frequency of 15 min every 2 h).
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Figure 3. The effect of the liquid culture system on the percentage share of individual developmental stages of Narcissus L. ‘Carlton’ embryos (early, mature and cotyledonary) in the total number of embryos obtained during multiplication and differentiation (Solid—solid medium (control); TIS 1 × 15—cultures in Rita® bioreactor with immersion frequency of 15 min every 24 h; RS 60/RS 100—cultures in liquid medium on rotary shaker (60 and 100 rpm).
Figure 3. The effect of the liquid culture system on the percentage share of individual developmental stages of Narcissus L. ‘Carlton’ embryos (early, mature and cotyledonary) in the total number of embryos obtained during multiplication and differentiation (Solid—solid medium (control); TIS 1 × 15—cultures in Rita® bioreactor with immersion frequency of 15 min every 24 h; RS 60/RS 100—cultures in liquid medium on rotary shaker (60 and 100 rpm).
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Table 1. Effect of immersion frequency on efficiency of Narcissus L. ‘Carlton’ repetitive somatic embryogenesis in Rita® bioreactor during multiplication and differentiation stages.
Table 1. Effect of immersion frequency on efficiency of Narcissus L. ‘Carlton’ repetitive somatic embryogenesis in Rita® bioreactor during multiplication and differentiation stages.
Immersion Frequency AMultiplicationDifferentiation
Biomass Growth
(6 Weeks)		No. of Somatic Embryos
(6 Weeks)		Biomass Growth
(6 Weeks)		No. of Somatic Embryos
(6 Weeks)
TIS 1 × 151.3 ± 0.2 d B51.7 ± 5.2 ab1.6 ± 0.7 ab152.6 ± 8.4 b
TIS 3 × 150.2 ± 0.0 a61.9 ± 6.9 b0.7 ± 0.2 a117.6 ± 5.9 a
TIS 12 × 150.4 ± 0.1 b41.8 ± 8.1 a1.6 ± 0.4 ab110.4 ± 27.2 a
Solid0.8 ± 0.0 c49.2 ± 7.3 a2.1 ± 0.7 b100.2 ± 8.5 a
Significant effects C************
A Immersion frequency: Solid—solid medium (control); TIS 1 × 15—cultures in bioreactor Rita® with immersion frequency of 15 min every 24 h; TIS 3 × 15—cultures in bioreactor Rita® with immersion frequency of 15 min every 8 h; TIS 12 × 15—cultures in bioreactor Rita® with immersion frequency of 15 min every 2 h. B Mean values ± SD followed by different lowercase letters are significantly different at p ≤ 0.05 according to Tukey’s multiple range test. C Significant effects: ***—at p ≤ 0.01.
Table 2. Effect of the liquid culture system used during multiplication and differentiation on the efficiency of Narcissus L. ‘Carlton’ repetitive somatic embryogenesis.
Table 2. Effect of the liquid culture system used during multiplication and differentiation on the efficiency of Narcissus L. ‘Carlton’ repetitive somatic embryogenesis.
Culture System During Multiplication (Mcs) ACulture System During Differentiation (Dcs)6th Week of Differentiation12th Week of Differentiation
Biomass Growth No. of Somatic Embryos Biomass GrowthNo. of Somatic Embryos
Effect of Mcs and Dcs
Solid Solid 2.2 ± 0.5 a–c B21.0 ± 1.3 ab4.7 ± 1.7 a–c38.5 ± 3.8 ab
SolidRS 605.9 ± 0.8 d14.1 ± 3.4 a11.7 ± 1.3 d41.0 ± 17.0 ab
RS 60Solid1.7 ± 0.2 ab24.0 ± 5.1 ab4.9 ± 1.0 a–c29.8 ± 4.5 a
RS 60RS 603.8 ± 0.8 c16.8 ± 3.2 a7.6 ± 0.6 c29.9 ± 9.0 a
RS 100Solid1.0 ± 0.3 a14.4 ± 10.2 a2.3 ± 0.5 a20.1 ± 12.1 a
RS 100RS 603.2 ± 1.0 bc17.0 ± 13.8 a6.2 ± 1.5 bc30.8 ± 17.2 a
TIS 1 × 15Solid1.5 ± 0.3 ab42.1 ± 7.7 b4.0 ± 0.2 ab65.1 ± 7.1 b
TIS 1 × 15RS 603.1 ± 0.3 bc22.2 ± 11.3 ab6.4 ± 0.6 bc44.9 ± 11.7 ab
Effect of Mcs
Solid 4.0 ± 2.1 b17.6 ± 4.4 a8.2 ± 4.1 c39.7 ± 11.1 ab
RS 602.7 ± 1.2 a20.3 ± 5.5 ab6.2 ± 1.7 b29.8 ± 6.4 a
RS 1002.1 ± 1.4 a15.7 ± 10.9 a4.3 ± 2.4 a25.4 ± 14.5 a
TIS 1 × 152.3 ± 0.9 a32.2 ± 13.9 b5.2 ± 1.4 ab55.0 ± 14.0 b
Effect of Dcs
Solid1.6 ± 0.5 a25.4 ± 12.2 b4.0 ± 1.4 a38.3 ± 18.7 a
RS 604.0 ± 1.3 b17.5 ± 8.4 a8.0 ± 2.5 b36.6 ± 13.8 a
Main effects C
Mcs × Dcs **ns***ns
Mcs ***********
Dcs ********ns
A Culture system: solid—solid medium (control); TIS 1 × 15—cultures in Rita® bioreactor with immersion frequency of 15 min every 24 h; RS60/RS100—cultures in liquid medium on rotary shaker (60 and 100 rpm). B Mean values ± SD followed by different lowercase letters are significantly different at p ≤ 0.05 according to Tukey’s multiple range test. C Main effects: ***—at p ≤ 0.01; **—at p ≤ 0.05; ns—not significant.
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Malik, M.; Tomiak, E.; Pawłowska, B. Effect of Liquid Culture Systems (Temporary Immersion Bioreactor and Rotary Shaker) Used During Multiplication and Differentiation on Efficiency of Repetitive Somatic Embryogenesis of Narcissus L. ‘Carlton’. Agronomy 2025, 15, 85. https://doi.org/10.3390/agronomy15010085

AMA Style

Malik M, Tomiak E, Pawłowska B. Effect of Liquid Culture Systems (Temporary Immersion Bioreactor and Rotary Shaker) Used During Multiplication and Differentiation on Efficiency of Repetitive Somatic Embryogenesis of Narcissus L. ‘Carlton’. Agronomy. 2025; 15(1):85. https://doi.org/10.3390/agronomy15010085

Chicago/Turabian Style

Malik, Małgorzata, Ewelina Tomiak, and Bożena Pawłowska. 2025. "Effect of Liquid Culture Systems (Temporary Immersion Bioreactor and Rotary Shaker) Used During Multiplication and Differentiation on Efficiency of Repetitive Somatic Embryogenesis of Narcissus L. ‘Carlton’" Agronomy 15, no. 1: 85. https://doi.org/10.3390/agronomy15010085

APA Style

Malik, M., Tomiak, E., & Pawłowska, B. (2025). Effect of Liquid Culture Systems (Temporary Immersion Bioreactor and Rotary Shaker) Used During Multiplication and Differentiation on Efficiency of Repetitive Somatic Embryogenesis of Narcissus L. ‘Carlton’. Agronomy, 15(1), 85. https://doi.org/10.3390/agronomy15010085

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