Protoplast Culture Research Articles

The cell colony development is connected with the accumulation of embryogenesis-related proteins and dynamic distribution of cell wall components in in vitro cultures of Fagopyrum tataricum and Fagopyrum esculentum

BackgroundDue to the totipotency of plant cells, which allows them to reprogram from a differentiated to a dedifferentiated state, plants exhibit a remarkable regenerative capacity, including under in vitro culture conditions. When exposed to plant hormones, primarily auxins and cytokinins, explant cells cultured in vitro can undergo differentiation through callus formation. Protoplast culture serves as a valuable research model for studying these processes in detail. This knowledge is particularly relevant for improving common and Tartary buckwheat species. To gain deeper insights into the stages of cell development from protoplasts—such as cell division, cell colony formation, and microcalli development—we focused on analyzing proteomes, cell wall composition, and changes in the expression profiles of selected genes in Fagopyrum protoplast cultures.ResultsThe results demonstrate a significant accumulation of somatic embryogenesis-related proteins like late embryogenesis abundant proteins (embryogenic protein-DC-8-like, seed biotin-containing protein) and endochitinases during the developmental path of protoplast-derived cultures. Additionally, we noted an extensive increase in seed storage proteins like vicilin, oleosins, and seed biotin-containing proteins during the culture. Investigation of somatic embryogenesis-associated transcription factors revealed massive up-regulation of LEAFY COTYLEDON1 for the 50th day of F. tataricum protoplast-derived cultures. However, for BABY BOOM, the transcription factor was noted to be down-regulated during the development of cell colonies. Furthermore, we demonstrated the variable distribution of cell wall components like pectin side chains, arabinogalactan proteins (AGPs) and extensins (EXTs), indicating the reorganisation of cell wall composition during the culture period.ConclusionsThis study revealed changes correlating with regaining embryogenic competence during the development of Fagopyrum protoplast-derived cell colonies. Our findings revealed variable expression levels of genes and proteins associated with somatic embryogenesis. This analysis identified an increase in seed storage proteins that play a significant role in the somatic somatic embryogenesis pathway of regeneration. Furthermore, the relationship between transcription factors and these processes seems to be connected with regaining somatic cells’ totipotency and promoting embryogenic competence of protoplast-derived cell colonies. Additionally, we observed dynamic changes in cell wall composition during the development of the protoplast-derived cultures.Clinical trial numberNot applicable.

A Comparison of DNA-Methylation during Protoplast Culture of Ponkan Mandarin (Citrus reticulata Blanco) and Tobacco (Nicotiana tabacum L.)

The epigenetic variation in protoplast regeneration is a topic that has attracted interest recently. To elucidate the role of DNA methylation in the regeneration of protoplasts from the ponkan (Citrus reticulata), this study employs the methylation-sensitive amplification polymorphism (MSAP) molecular marker technique to analyze changes in DNA methylation levels and patterns during the isolation and culture of protoplasts from ponkan and tobacco. Additionally, differential DNA methylation fragments are cloned, sequenced, and subjected to bioinformatics analysis. The results reveal that, for non-regenerable ponkan mesophyll protoplasts, DNA methylation levels increase by 3.98% after isolation and then show a trend of initial decrease followed by an increase during culture. In contrast, for regenerable ponkan callus protoplasts and tobacco mesophyll protoplasts, DNA methylation levels decrease by 1.75% and 2.33%, respectively, after isolation. During culture, the DNA methylation levels of ponkan callus protoplasts first increase and then decrease, while those of tobacco mesophyll protoplasts show an opposite trend of initial decrease followed by an increase. Regarding DNA methylation patterns, ponkan mesophyll protoplasts exhibit primarily hypermethylation changes accompanied by a small amount of gene demethylation, whereas ponkan callus protoplasts are dominated by demethylation changes with some genes undergoing hypermethylation. The methylation exhibits dynamic changes in protoplast isolation regeneration. By recovering, cloning, sequencing, and performing BLASTn alignment analysis on specific methylation modification sites in the ponkan, 18 DNA sequences with high homology are identified which are found to be involved in various biological functions, thereby establishing a foundational basis for genetic editing in protoplasts.

Small molecule inhibitors of human LRRK2 enhance in vitro embryogenesis and microcallus formation for plant regeneration of crop and model species

In vitro plant embryogenesis and microcallus formation are systems which are required for plant regeneration, a process during which cell reprogramming and proliferation are critical. These systems offer many advantages in breeding programmes, such as doubled-haploid production, clonal propagation of selected genotypes, and recovery of successfully gene-edited or transformed plants. However, the low proportion of reprogrammed cells in many plant species makes these processes highly inefficient. Here we report a new strategy to improve in vitro plant cell reprogramming using small molecule inhibitors of mammalian leucine rich repeat kinase 2 (LRRK2), which are used in pharmaceutical applications for cell reprogramming, but never used in plants before. LRRK2 inhibitors increased in vitro embryo production in three different systems and species, microspore embryogenesis of oilseed rape and barley, and somatic embryogenesis in cork oak. These inhibitors also promoted plant cell reprogramming and proliferation in Arabidopsis protoplast cultures. The benzothiazole derivative JZ1.24, a representative compound of the tested molecules, modified the expression of the brassinosteroid (BR)-related genes BIN2, CPD, and BAS1, correlating with an activation of BR signaling. Additionally, the LRRK2 inhibitor JZ1.24 induced the expression of the embryogenesis marker gene SERK1-like. The results suggest that the use of small molecules from the pharmaceutical field could be extended to promote in vitro reprogramming of plant cells towards embryogenesis or microcallus formation in a wider range of plant species and in vitro systems. This technological innovation would help to develop new strategies to improve the efficiency of in vitro plant regeneration, a major bottleneck in plant breeding.

High Efficiency of the Somatic Embryogenesis Sequences of Desert Lime (Eremocitrus Glauca)

Abstract: Eremocitrus glauca is a desert plant that is sources to the plant genetic resources for citrus, because these plants are resistant cultivars to some enemy mainly in the tropical region. Isolated protoplast culture media supplemented with to 2-isopentenyladenine (2iP) maintaining under the light intensity range 13.23. mol/m2/s1 to 70.8 mol/m2/s1 sigificantly effect to plating effisiensy protoplasts, globular somatic embryos, and plant regeneration of Eremocitrus glauca. The temperature emission and light illumination are applicate to cells has implicated to cell recovery and formation of somatic embryos. 2iP and environment factors increased the health cell from enzyms stress, grow and developed rapidly. In vitro temperature for somatic embryo formation in cultures is achieved on 25oC in corporations with 0.1 mg/l, 2 iP and more likely in pattern linear, with equation Y = 0,7336x + 4,1894, R2 = 0.8172. The same patern is also accur to ligh illumination. Attractive conclusion that light and temperature must be well to do on plant physiology and chemistry. The sequence from isolated protoplast to cell recovery, formation of somatic embryos and plant or shoot regeneration under control of media, plant growht regulators and environment factors has establish in Eremocitrus glauca. Forturmore the gene tranfer from this species has been easily.

Response surface methodology mediated optimization of phytosulfokine and plant growth regulators for enhanced protoplast division, callus induction, and somatic embryogenesis in Angelica Gigas Nakai

BackgroundAngelica Gigas (Purple parsnip) is an important medicinal plant that is cultivated and utilized in Korea, Japan, and China. It contains bioactive substances especially coumarins with anti-inflammatory, anti-platelet aggregation, anti-cancer, anti-diabetic, antimicrobial, anti-obesity, anti-oxidant, immunomodulatory, and neuroprotective properties. This medicinal crop can be genetically improved, and the metabolites can be obtained by embryonic stem cells. In this context, we established the protoplast-to-plant regeneration methodology in Angelica gigas.ResultsIn the present investigation, we isolated the protoplast from the embryogenic callus by applying methods that we have developed earlier and established protoplast cultures using Murashige and Skoog (MS) liquid medium and by embedding the protoplast in thin alginate layer (TAL) methods. We supplemented the culture medium with growth regulators namely 2,4-dichlorophenoxyaceticacid (2,4-D, 0, 0.75, 1.5 mg L− 1), kinetin (KN, 0, 0.5, and 1.0 mg L− 1) and phytosulfokine (PSK, 0, 50, 100 nM) to induce protoplast division, microcolony formation, and embryogenic callus regeneration. We applied central composite design (CCD) and response surface methodology (RSM) for the optimization of 2,4-D, KN, and PSK levels during protoplast division, micro-callus formation, and induction of embryogenic callus stages. The results revealed that 0.04 mg L− 1 2,4-D + 0.5 mg L− 1 KN + 2 nM PSK, 0.5 mg L− 1 2,4-D + 0.9 mg L− 1 KN and 90 nM PSK, and 1.5 mg L− 1 2,4-D and 1 mg L− 1 KN were optimum for protoplast division, micro-callus formation and induction embryogenic callus. MS basal semi-solid medium without growth regulators was good for the development of embryos and plant regeneration.ConclusionsThis study demonstrated successful protoplast culture, protoplast division, micro-callus formation, induction embryogenic callus, somatic embryogenesis, and plant regeneration in A. gigas. The methodologies developed here are quite useful for the genetic improvement of this important medicinal plant.

Effects of Salts on Helical Protoplast-Callose-Fiber Formation and Cell Division in Leaf Protoplast Culture of Arabidopsis thaliana: Ultrastructure of PCF Using Transmission Electron Microscopy

Effects of four salts addition (10-200 mM), NaCl, KCl, MgCl2, and CaCl2 were examined on the growth of protoplasts and formation of helical 1.5 mm long, protoplast-callose-fibers (PCF) in Arabidopsis thaliana liquid leaf protoplast cultures. The basal medium was Murashige and Skoog’s medium containing 1 μM 2,4-dichlorophenoxyacetic acid and 0.1 μM benzyladenine, 3% sucrose, and 0.4 M mannitol. The protoplast division was highly stimulated by the addition of 50 mM Ca2+ ion but was totally inhibited by 50 mM Mg2+ ion. Inhibition by K+ and Na+ ions was in-between. By contrast, PCF formation, whose numbers were counted under a fluorescence inverted microscope after Aniline Blue staining for β-1,3-glucan (callose), was stimulated by both K+ and Ca2+ ions but inhibited by Mg2+ ion. After selecting Arabidopsis PCF using a micromanipulator, the sub-fibril ultrastructure was examined by transmission electron microscopy (TEM). The PCFs of Betula platyphylla and Larix leptolepis, cultured with 200 mM Ca2+ and 50 mM Mg2+ ions, respectively, after long-term storage and rehydration, were examined by TEM. The effects of different factors were discussed on PCF formation and sub-structures in different herbaceous and tree plant species.

Isolation, culture of Platycodon grandiflorus protoplasts: factors affecting protoplast yield, cell division, and micro-callus formation

Isolation, culture of Platycodon grandiflorus protoplasts: factors affecting protoplast yield, cell division, and micro-callus formation

Plant improvement and metabolite production in Cannabis sativa: Recent biotechnological advances

The Cannabis sativa plant is an excellent source of metabolites, fiber, and medicinal properties. Phytocannabinoids are the secondary metabolites naturally derived from Cannabis plant species. These metabolites are promising and can be used in producing phytomedicine or plant-based therapeutics. However, many of these compounds are produced in low quantities across different Cannabis species. To solve this limitation, in vitro, biotechnological methods offer promising solutions for enhancing the production of secondary metabolites in Cannabis. This review highlights the biotechnological approaches for enhancing Cannabis secondary metabolite production through in vitro plant improvement techniques such as plant regeneration, elicitor-responsive metabolite induction, polyploidy manipulation, protoplast culture, bioreactor-based hairy root culture, genetic transformation, and genome editing. These biotechnological approaches might be useful for improving Cannabis plants and increasing plant capacity to produce potential metabolites. These phytochemical and bioactive compounds found in Cannabis species could be used as alternative resources for pharmaceutical and industrial production.

Protoplast Isolation, Culture, and Regeneration in Common and Tartary Buckwheat.

Techniques based on the use of plant protoplasts are a convenient model for better understanding and observing developmental changes in the cells. The establishment of research tools based on protoplasts consists of many steps needed for optimization. Here, we describe the culture of morphogenic callus (MC)- and hypocotyl-derived protoplasts of common (Fagopyrum esculentum Moench) and Tartary (F. tataricum (L.) Gaertn.) buckwheat. Protoplasts embedding in agarose matrix and application of plant hormones, including phytosulfokine (PSK), enable the development of protoplast cultures and plant regeneration.

Immunodetection of Cell Wall Components in Studies on Cell Wall Rebuilding in Fagopyrum esculentum and Fagopyrum tataricum.

Immunocytochemical studies of the cell wall are used to visualize specific epitopes of pectins, arabinogalactan proteins, hemicelluloses, extensins, and other wall components using specific primary antibodies. This reaction, combined with calcofluor staining, allows to comprehend how the cell wall is rebuilt during the protoplast culture. In this protocol, the method of immunostaining using antibodies against cell wall components based on Fagopyrum esculentum and Fagopyrum tataricum protoplasts is described.

2 in 1 Vectors Improve in Planta BiFC and FRET Analysis.

Protein-protein interactions (PPIs) play vital roles in all subcellular processes, and a number of tools have been developed for their detection and analysis. Each method has its unique set of benefits and drawbacks that need to be considered prior application. In fact, researchers are spoilt for choice when it comes to deciding which method to use for the initial detection of a PPI and which to corroborate the findings. With constant improvements in microscope development, the possibilities of techniques to study PPIs in vivo, and in real time, are continuously enhanced and expanded. Here, we describe three common approaches, their recent improvements incorporating a 2-in-1 cloning approach, and their application in plant cell biology: ratiometric bimolecular fluorescence complementation (rBiFC), FRET acceptor photobleaching (FRET-AB), and fluorescent lifetime imaging (FRET-FLIM), using Nicotiana benthamiana leaves and Arabidopsis thaliana cell culture protoplasts as transient expression systems.

Isolation, culture of protoplasts of Angelica gigas Nakai and regeneration of plants via somatic embryogenesis

Isolation, culture of protoplasts of Angelica gigas Nakai and regeneration of plants via somatic embryogenesis

An efficient protoplast-based genome editing protocol for Vitis species.

CRISPR-Cas technologies allow for precise modifications in plant genomes and promise to revolutionize agriculture. These technologies depend on the delivery of editing components into plant cells and the regeneration of fully edited plants. In vegetatively propagated plants, such as grape, protoplast culture provides one of the best avenues for producing non-chimeric and transgene-free genome-edited plants. However, poor regeneration of plants from protoplasts has hindered their implementation for genome editing. Here, we report an efficient protocol for regenerating plants from protoplasts from multiple grape varieties. By encapsulating the protoplasts in calcium alginate beads and co-culturing them with feeder cultures, the protoplasts divide to form callus colonies that regenerate into embryos and ultimately plants. This protocol worked successfully in wine and table grape (Vitis vinifera) varieties, as well as grape rootstocks and the grapevine wild relative Vitis arizonica. Moreover, by transfecting protoplasts with CRISPR-plasmid or ribonucleoprotein (RNP) complexes, we regenerated albino plants with edits in VvPHYTOENE DESATURASE gene in three varieties and in V. arizonica. The results reveal the potential of this platform to facilitate genome editing in Vitis species.

Plant Extracellular Vesicles: Current Landscape and Future Directions.

Plant cells secrete membrane-enclosed micrometer- and nanometer-sized vesicles that, similarly to the extracellular vesicles (EVs) released by mammalian or bacterial cells, carry a complex molecular cargo of proteins, nucleic acids, lipids, and primary and secondary metabolites. While it is technically complicated to isolate EVs from whole plants or their tissues, in vitro plant cell cultures provide excellent model systems for their study. Plant EVs have been isolated from the conditioned culture media of plant cell, pollen, hairy root, and protoplast cultures, and recent studies have gathered important structural and biological data that provide a framework to decipher their physiological roles and unveil previously unacknowledged links to their diverse biological functions. The primary function of plant EVs seems to be in the secretion that underlies cell growth and morphogenesis, cell wall composition, and cell-cell communication processes. Besides their physiological functions, plant EVs may participate in defence mechanisms against different plant pathogens, including fungi, viruses, and bacteria. Whereas edible and medicinal-plant-derived nanovesicles isolated from homogenised plant materials ex vivo are widely studied and exploited, today, plant EV research is still in its infancy. This review, for the first time, highlights the different in vitro sources that have been used to isolate plant EVs, together with the structural and biological studies that investigate the molecular cargo, and pinpoints the possible role of plant EVs as mediators in plant-pathogen interactions, which may contribute to opening up new scenarios for agricultural applications, biotechnology, and innovative strategies for plant disease management.

Early selection of carrot somatic hybrids: a promising tool for species with high regenerative ability

BackgroundSince its discovery, somatic hybridization has been used to overcome the sexual barriers between cultivated and wild species. A combination of two somatic cells might provide a novel set of features, often of agronomical importance. Here, we report a successful approach for production and selection of interspecific somatic hybrid plants between cultivated and wild carrot using dual-labelling of protoplasts and an early selection of fused cells via micromanipulator. Both subspecies used in this study are characterised by a very high regenerative ability in protoplast cultures. Thus, a precise and effective method of hybrid selection is essential to assure the development and regeneration of much less numerous heterokaryons in the post-fusion cell mixture.ResultsElectrofusion parameters, such as alternating current and direct current, were optimised for an efficient alignment of protoplasts and reversible membrane breakdown followed by a cell fusion. Four hundred twenty-nine cells emitting green–red fluorescence, identified as hybrids, were obtained. Co-culture with donor-derived protoplasts in the alginate feeder layer system stimulated re-synthesis of the cell wall and promoted cell divisions of fusants. Somatic embryogenesis occurred in hybrid-derived microcalli cultures, followed by plant regeneration. Regenerated hybrids produced yellowish storage roots and leaves of an intermediate shape between cultivated and wild subspecies. The intron length polymorphism analysis revealed that 123 of 124 regenerated plants were hybrids.ConclusionsThe developed protocol for protoplast fusion and an early selection of hybrids may serve as an alternative to combining genomes and transferring nuclear or cytoplasmatic traits from wild Daucus species to cultivated carrot.

Optimization of Protoplast Preparation System from Leaves and Establishment of a Transient Transformation System in Apium graveolens

Protoplast culture and transformation technology offer a novel method for developing new plant varieties. Nonetheless, the effective preparation of protoplasts and transformation technology specific to celery has yet to be achieved. This study utilized celery seedling leaves as the primary materials to examine the key factors influencing protoplast isolation. The aim was to prepare leaf protoplasts with a high yield and of high quality and subsequently conduct transient gene transformation and expression. The findings indicated that the most effective procedure for isolating and purifying protoplasts was enzymatic digestion using an enzyme solution consisting of 2.0% cellulase, 0.1% pectolase, and 0.6 M mannitol for a duration of 8 h. Subsequently, the protoplasts were filtered through a 400-mesh sieve and purified through centrifugation at 200× g. Within this system, the overall protoplast yield was exceptionally high, reaching a viability rate of up to 95%. The transient transformation system yielded a maximum transformation efficiency of approximately 53%, as evaluated using the green fluorescent protein (GFP) as a reporter gene. The parameters of the transient transformation system were as follows: a protoplast concentration of 5 × 105 cells·mL−1, exogenous DNA concentration of 500 μg·mL−1, final concentration of PEG4000 at 40%, and transformation duration of 15 min. The transient transformation system was also utilized to further analyze the protein localization characteristics of the celery transcription factor AgMYB80. The findings indicated that AgMYB80 predominantly localizes in the nucleus, thereby confirming the reliability and effectiveness of the transient transformation system. This study successfully established an efficient system for isolating, purifying, and transforming celery protoplasts, and will serve as a basis for future studies on molecular biology and gene function.

Promotive effect of phytosulfokine - peptide growth factor - on protoplast cultures development in Fagopyrum tataricum (L.) Gaertn

BackgroundFagopyrum tataricum (Tartary buckwheat) is a valuable crop of great nutritional importance due to its high level of bioactive compounds. Excellent opportunities to obtain plants with the high level or the desired profile of valuable metabolites may be provided by in vitro cultures. Among known in vitro techniques, protoplast technology is an exciting tool for genetic manipulation to improve crop traits. In that context, protoplast fusion may be applied to generate hybrid cells between different species of Fagopyrum. To apply protoplast cultures to the aforementioned approaches in this research, we established the protoplast-to-plant system in Tartary buckwheat.ResultsIn this work, cellulase and pectinase activity enabled protoplast isolation from non-morphogenic and morphogenic callus (MC), reaching, on average, 2.3 × 106 protoplasts per g of fresh weight. However, to release protoplasts from hypocotyls, the key step was the application of driselase in the enzyme mixture. We showed that colony formation could be induced after protoplast embedding in agarose compared to the alginate matrix. Protoplasts cultured in a medium based on Kao and Michayluk supplemented with phytosulfokine (PSK) rebuilt cell walls, underwent repeated mitotic division, formed aggregates, which consequently led to callus formation. Plating efficiency, expressing the number of cell aggregate formed, in 10-day-old protoplast cultures varied from 14% for morphogenic callus to 30% for hypocotyls used as a protoplast source. However plant regeneration via somatic embryogenesis and organogenesis occurred only during the cultivation of MC-derived protoplasts.ConclusionsThis study demonstrated that the applied protoplast isolation approach facilitated the recovery of viable protoplasts. Moreover, the embedding of protoplasts in an agarose matrix and supplementation of a culture medium with PSK effectively stimulated cell division and further development of Tartary buckwheat protoplast cultures along with the plant regeneration. Together, these results provide the first evidence of developing a protoplast-to-plant system from the MC of Fagopyrum tataricum used as source material. These findings suggest that Tartary buckwheat’s protoplast cultures have potential implications for the species’ somatic hybridization and genetic improvement.

Efficient and rapid system of plant regeneration via protoplast cultures of Fagopyrum esculentum Moench

In the present study, a high yield of isolated protoplasts from the agronomically important crop Fagopyrum esculentum was obtained by applying a mixture of cellulase, pectolyase, and driselase. We demonstrated that the yield of morphogenic callus-derived protoplasts was 1 × 106 protoplasts per g of fresh tissue. For hypocotyls used as the protoplast source, the number of released cells was twice lower. The protoplasts, embedded in an agarose matrix and cultured in a modified Kao and Michayluk media supplemented with phytosulfokine, re-enter the cell cycle and start to develop, forming microcalli. The plating efficiency was about 20% in the case of hypocotyl- and morphogenic callus-derived protoplasts. For plant regeneration, the medium was supplemented with different combinations of cytokinin. Somatic embryogenesis and organogenesis occur during the cultivation of the protoplast-derived tissues, depending on the applied protoplast source. For the first time, an effective protoplast-to-plant system for F. esculentum has been developed.

Effects of TSA, NaB, Aza in Lactuca sativa L. protoplasts and effect of TSA in Nicotiana benthamiana protoplasts on cell division and callus formation.

Whole-plant regeneration via plant tissue culture is a complex process regulated by several genetic and environmental conditions in plant cell cultures. Recently, epigenetic regulation has been reported to play an important role in plant cell differentiation and establishment of pluripotency. Herein, we tested the effects of chemicals, which interfere with epigenetic regulation, on the plant regeneration from mesophyll protoplasts of lettuce. The used chemicals were histone deacetylase inhibitors trichostatin A (TSA) and sodium butyrate (NaB), and the DNA methyltransferase inhibitor azacytidine (Aza). All three chemicals increased cell division, micro-callus formation and callus proliferation in lettuce protoplasts. Cell division increased by more than 20% with an optimal treatment of the three chemicals. In addition, substantial increase in the callus proliferation rates was observed. In addition, TSA enhances cell division and adventitious shoot formation in the protoplast culture of Nicotiana benthamiana. The regenerated tobacco plants from TSA-treated protoplasts did not show morphological changes similar to the control. TSA increased histone H3 acetylation levels and affected the expression of CDK, CYCD3-1, and WUS in tobacco protoplasts. Thus, we investigated the effect of TSA, NaB, and Aza on Lactuca sativa L. protoplasts and the effect of TSA on cell division and callus formation in Nicotiana benthamiana protoplasts, which facilitates plant regeneration from mesophyll protoplasts. Furthermore, these chemicals can be directly applied as media additives for efficient plant regeneration and crop improvement in various plant species.

Plant regeneration from protoplasts of Pastinaca sativa L. via somatic embryogenesis

In the present study we report the development of an effective and relatively efficient protocol for protoplast-to-plant regeneration of parsnip (Pastinaca sativa L.) via indirect somatic embryogenesis. The regenerative potential of three open-pollinated and four hybrid cultivars was assessed. The protoplast isolation efficiency after digestion of source material in an enzyme mixture consisted of 1% cellulase Onozuka R-10 and 0.1% pectolyase Y-23 reached on average 3.6 × 106 of cells per g of fresh mass. Protoplasts embedded in an alginate matrix and cultured in parsnip protoplast culture medium with phytosulfokine-α and putrescine reconstructed their cell wall and re-entered mitotic divisions. After the release from alginate, microcallus proliferated continuously on Gamborg B5 medium with vitamins supplemented with 100 nM of phytosulfokine-α. Indirect somatic embryogenesis occurred during the callus culture of cultivar ‘Półdługi biały’. The regenerated and acclimatized plants were morphologically similar to their donors and displayed no variation in the ploidy level.