Rapid propagation of plants by tissue culture is of great significance for large-scale production, molecular genetics research, and breeding. Currently, a rapid and high-efficient tissue culture protocol for Euonymus bungeanus is needed. To develop a propagation system for this species, we established a new regeneration system from leaves. Callus formation was induced on Murashige and Skoog (MS) medium supplemented with 0.5 mg dm^-3 6-benzylaminopurine (6-BA) and 0.5 mg dm^-3 α-naphthalene acetic acid (NAA) and the induction rate almost reached 100 % under red radiation within 21 d. The medium for proliferation of adventitious buds comprised of MS medium with 1.0 mg dm^-3 6-BA and 0.5 mg dm^-3 NAA, and the induction rate within 20 d nearly reached 100 %. When, the adventitious buds were transferred to the rooting medium containing 1/2MS, 2.0 mg dm^-3 indole-3-butyric acid (IBA), and 0.05 mg dm^-3 NAA, adventitious root formation was achieved within 20 d. Collectively, the rapid and high-efficient regeneration system from E. bungeanus leaves was established, providing useful references for effective mass propagation and it could serve as an enabling technology for future genetic engineering.

An efficient plant regeneration protocol has been established for two commercial Populus hybrid clones, MC (Populus × euramericana) and UNAL (Populus × interamericana). The culture of internode segments on Murashige and Skoog (MS) medium with 0.5 μM α-naphthalene acetic acid (NAA) and 4 μM N⁶-benzyladenine for 7 weeks (2 weeks in absence of activated charcoal and 5 weeks in its presence) resulted in the highest frequency of shoot regeneration (100 % for MC and 82 % for UNAL). All regenerated shoots longer than 2 cm rooted on half-strength MS medium, independent of the addition of 0.1 μM NAA. Nevertheless, shoots developed better-formed roots in NAA-free medium, which had a positive effect on the acclimatization of plants. In order to know the cellular processes underlying in vitro shoot organogenesis, a histological study was made in UNAL internode-explants. Results revealed that in vitro culture caused swelling around the cut-off zones in all explants, but only those undergoing organogenesis formed proliferation centers under subepidermal cells, which led to formation of bud primordia. Moreover, in vivo tissues and explants with different in vitro response showed different immunolabelling patterns when they were treated with fluorescentmonoclonal antibodies directed to several pectin-polysaccharides of the cell wall. Results allow us to assign a predominant role of homogalacturonan with a low degree of methyl-esterification in the initiation of bud primordia, role of β-1,4-D-galactan side chains of rhamnogalacturonan-I in the cellular differentiation, role of α-1,5-L-arabinan side chains of rhamnogalacturonan-I and of homogalacturonan with a high degree of methyl-esterification in cell division and growth.

Plant regeneration through indirect somatic embryogenesis was attempted from the immature cotyledon-derived explant of Cassia angustifolia Vahl. - a valuable leguminous shrub. The highest frequency (90.5 %) of somatic embryos was obtained on a Murashige and Skoog (MS) medium augmented with 10.0 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.0 μM benzyladenine (BA) with the production of a maximum of 22.8 embryos per explant, of which 35.3 % germinated on the same medium after 6 weeks of culture. A half strength MS medium without plant growth regulators facilitated better conversion of embryos into complete plantlets compared to a full strength MS medium. Regenerated plantlets were successfully acclimatized in sterile Soilrite and transferred to field conditions with a 70 % survival rate. Histological studies performed at different stages of embryogenesis revealed the mode of differentiation of embryos from the callus. The content of chlorophylls (a + b) and carotenoids, and the net photosynthetic rate (P_N) in the regenerated plantlets were tested during different periods of acclimatization.

An efficient in vitro regeneration protocol through somatic embryogenesis was established from stem transverse thin cell layers (tTCLs) of Dendrobium aqueum Lindley, an imperiled orchid. This study outlines the induction and successive maturation stages of D. aqueum somatic embryos (SEs). The tTCLs (~ 0.5 mm thick) cultured on halfstrength Murashige and Skoog (MS) medium containing cytokinins and auxins, either individually or in combination, produced embryogenic callus (EC). Treatment with 0.5 mg dm^-3 zeatin induced EC in 41.42 % of tTCLs. As many as 42.66 globular SEs per tTCL were formed in the presence of 1.5 mg dm^-3N⁶-(2-isopentyl) adenine (2iP) but only on 10.33 % of explants. The combined treatment of 2iP (1.5 mg dm^-3) and 0.5 mg dm^-3 6-benzyladenine resulted in 34 globular SEs on 14.7 % of tTCLs whereas the combination of 2iP and 1.0 mg dm^-3 indole-3-butyric acid (IBA) induced 7.4 globular SEs on 52.33 % of tTCLs. Supplementation of activated charcoal, amino acids, and antioxidants alleviated browning at all the concentrations tested, but the EC response declined. The addition of 0.5 mg dm^-3 polyvinylpyrrolidone to 1.5 mg dm^-3 2iP and 1.0 mg dm^-3 IBA produced 24 SEs on 19.89 % of tTCLs suggesting that the EC and SEs can be effectively induced by individual cytokinins whereas the synergistic treatments with other compounds can only enhance the induction of EC. Histological observations of EC showed the formation of globular SEs from sub-epidermal regions. Successive developmental stages of globular SEs and the intermediate stage of protocorm like bodies until the formation of plantlets were observed. The plantlets obtained through SEs showed no morphological variations, and inter simple sequence repeat profiles also confirmed the genetic fidelity of in vitro-derived progeny with high monomorphism (97.78 %). In conclusion, the use of stem tTCLs is an effective method to produce SEs through indirect somatic embryogenesis in D. aqueum.

Over the past few decades, rice (Oryza sativa L.) has remained a fundamental staple crop and a primary nutritional energy source for nearly 3.5 billion people worldwide, particularly in Asia. With the global population projected to reach 9.6 billion by 2050, rice production must significantly increase to meet the escalating food demand. However, salinity stress poses a major abiotic challenge that severely hampers plant growth and productivity. Soil salinization, driven by climate change and rising temperatures, leads to an excessive accumulation of salts in the soil (Sári et al., 2023). This phenomenon disrupts plant physiology through water deficit, cytotoxic effects of Na⁺ and Cl⁻ ion accumulation, and nutrient imbalances (Isayenkov and Maathuis, 2019). In coastal regions, salinity stress is further intensified by seawater intrusion into groundwater reserves (Muhardi et al., 2020), while in arid and semi-arid areas, low rainfall limits salt leaching, resulting in excessive salt accumulation (Karolinoerita and Yusuf, 2020). Exposure to salinity stress induces the overproduction of reactive oxygen species (ROS), a group of highly reactive free radicals that can damage essential cellular components, including DNA, proteins, lipids, and pigments, ultimately impairing plant function (Ghosh et al., 2021). To mitigate these detrimental effects, plants activate various adaptive responses (Huong et al., 2020), including the upregulation of antioxidant enzyme systems (Jan et al., 2019), which play a crucial role in ROS scavenging and oxidative stress alleviation. These responses involve both well-developed enzymatic and non-enzymatic scavenging pathways or detoxification systems to counter the destructive effects of ROS that include the enzymes superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR), and so forth (Hasanuzzaman et al., 2011).

Alternative respiration pathway (AP) is an important pathway which can be induced by environment stresses in plants. In the present study, we show a new mechanism involving the AP in nitrogen deprivation-induced tolerance of Poa annua callus to salt stress. The AP capacity markedly increased under a 600 mM NaCl treatment or nitrogen deprivation pretreatment and reached a maximum under the nitrogen deprivation pretreatment combined with the NaCl treatment (-N+NaCl). Malondialdehyde (MDA) and H₂O₂ content and Na⁺/K⁺ ratio significantly increased under the 600 mM NaCl treatment but less under the-N+NaCl treatment. Moreover, both the nitrogen deprivation and the NaCl stress stimulated the plasma membrane (PM) H⁺-ATPase activity and increased pyruvate content. The maximal stimulating effect was found under the-N+NaCl treatment. When the AP capacity was reduced by salicylhydroxamic acid (SHAM, an inhibitor of AP), content of MDA and H₂O₂ and Na⁺/K⁺ ratio dramatically increased, whereas PM H⁺-ATPase activity decreased. Moreover, exogenous application of pyruvate produced a similar effect as the nitrogen deprivation pretreatment. The effects of SHAM on the Poa annua callus were counteracted by catalase (a H₂O₂ scavenger) and diphenylene iodonium (a plasma membrane NADPH oxidase inhibitor). Taken together, our results suggest that the nitrogen deprivation enhanced the capacity of AP by increasing pyruvate content, which in turn prevented the Poa annua callus from salt-induced oxidative damages and Na⁺ over-uptake.

Ascorbic acid (AsA) is naturally occurring compound with antioxidant activity and plays a pivotal role in plant cell adaptation to salinity stress. The objective of this work was to assess the influence of exogenous AsA on the embryogenic callus of indica rice (Oryza sativa L.) cv. MRQ74 cultivated under saline conditions. NaCl (200 mM) decreased callus fresh and dry masses, relative growth rate, and K⁺ and Ca⁺² content, and increased Na⁺ content and Na⁺/K⁺ ratio. Application of AsA (0.5 or 1 mM) alleviated these effects of salinity. Activities of peroxidase, catalase, superoxide dismutase, as well as content of proline increased due to the NaCl treatment, and these parameters were mostly further increased by 0.5 mM AsA. Thus, AsA can increase callus tolerance to NaCl stress.

The utility of plant tissue culture for the mass propagation of trees is well known, but continuous in vitro multiplication of plant material may increase the possibility of somaclonal variation; therefore, it is essential to evaluate the genetic integrity of regenerants from species-specific in vitro protocols prior to mass production and implementation. The objectives of this study were: 1) to determine the effect of 2,4-dichlorophenoxyacetic acid (2,4-D) concentration over two cycles of secondary somatic embryogenesis in Magnolia dealbata; and 2) to verify the genetic stability of the regenerants obtained. The embryogenic response was not significantly affected by the concentration of 2,4-D but did vary across cycles of induction. The addition of 4.52 μM 2,4-D induced the highest total number of embryos (100.5), the mean number of somatic embryos (25.1) and somatic embryos per explant (80.6). In both 2,4-D concentration (2.26 or 4.52 μM), genetic integrity between the donor and the propagated clones was 0.90, and the low genetic instability (≤ 0.10 in both PGR treatments) might be due to effect of cyclic somatic embryogenesis or the different response of the explants at stress in in vitro culture conditions. However, it is necessary to examine more cell lines and somatic embryogenesis cycles.

An in vitro propagation system was developed for castor-bean (Ricinus communis L. cv. TMV 6) through cotyledon derived callus cultures. The impact of different concentrations of auxins, cytokinins, additives, amino acids and sugars were evaluated for callus induction and shoot proliferation. Green compact nodular organogenic callus was obtained on the medium fortified with Murashige and Skoog (MS) salts, B5 vitamins, 2.0 mg dm^-3 6-benzyladenine and 0.8 mg dm^-3 α-naphthalene acetic acid (NAA). Multiple shoot proliferation from the callus cultures was achieved on the medium with MS salts, B5 vitamins, 2.5 mg dm^-3 thidiazuron (TDZ), 0.4 mg dm^-3 NAA and 15 mg dm^-3 glutamine. During multiple shoot induction the phenolic secretion was controlled by the addition of 15 mg dm^-3 polyvinylpyrolidone. The proliferated shoots were elongated on the medium comprising MS salts, B5 vitamins, 1.5 mg dm^-3 TDZ and 0.3 mg dm^-3 gibberellic acid. The elongated shoots were rooted on the medium containing MS salts, B5 vitamins, 0.3 mg dm^-3 indole-3-butyric acid and 0.6 mg dm^-3 silver nitrate. After root induction, the plants were hardened in earthen pots containing sand, soil and vermiculite.

Tissue culture of hazelnut (Corylus avellana L.) represented the promising strategy for production of its valuable compound, paclitaxel, but one of the most important problems is initial callus browning in its callus culture. To obtain healthy callus cells of C. avellana, three different culture media, Murashige and Skoog (MS), Nas and Read (NRM), and Driver and Koniuki (DKW), in combination with 500 mg dm^-3 citric acid, 500 mg dm^-3 acetic acid, and 500 mg dm^-3 polyvinylpyrrolidone (PVP) were applied. These cultures were passed two sub-cultures in the same media. Also, a novel modified sub-culture system was designed and compared to routine techniques. The sub-culture system was changed as following: the induced calli were transferred to a liquid media with the same composition and after developing the cell suspension, the cells were immobilized on a solid medium. In the first experiment, it was demonstrated that the medium compositions had an effect on the growth rate and callus browning reduction although they could not eliminate the browning. The results have shown that the highest growth indices were related to NRM with 500 mg dm^-3 acetic acid + 500 mg dm^-3 citric acid and to NRM with 500 mg dm^-3 PVP in the first culture. These media had the least amount of browning (9.15 %). In the two next sub-cultures, NRM without additives had the maximum growth rate and the lowest browning was observed on the same media as in the first subculture (13.3 %). The technique of modified sub-cultures increases growth rate ten times in comparison with routine cultivation method.

In the present study, in vitro regeneration system for a recalcitrant woody tree legume, Leucaena leucocephala (cvs. K-8, K-29, K-68 and K-850) from mature tree derived nodal explants as well as seedling derived cotyledonary node explants was developed. Best shoot initiation and elongation was found on full-strength Murashige and Skoog (MS) medium supplemented with 3 % (m/v) sucrose, 100 mg dm^-3 myoinositol, 100 mg dm^-3 glutamine, 20.9 µM N ⁶-benzylamino-purine (BAP) and 5.37 µM 1-naphthalene acetic acid (NAA). Rooting was induced in half-strength MS medium containing 2 % (m/v) sucrose, 100 mg dm^-3 myoinositol, 14.76 µM indole-3-butyric acid (IBA) and 0.23 µM kinetin. The cultivar K-29 gave the best response under in vitro conditions. Rooted plantlets were subjected to hardening and successfully transferred to greenhouse. Further, somatic embryogenesis from nodal explants of cv. K-29 via an intermittent callus phase was also established. Pronounced callusing was observed on full-strength MS medium containing 3 % (m/v) sucrose, 100 mg dm^-3 myoinositol, 40.28 µM NAA and 12.24 µM BAP. These calli were transferred to induction medium and maximum number of globular shaped somatic embryos was achieved in full-strength MS medium fortified with 3 % (m/v) sucrose, 100 mg dm^-3 myoinositol, 15.0 µM 2,4-dichlorophenoxyacetic acid (2,4-D), 5.0 µM BAP and 1.0 mM proline. Moreover, an increase in endogenous proline content up to 28^th day of culture in induction medium was observed. These globular shaped somatic embryos matured in full-strength MS medium with 3 % (m/v) sucrose, 100 mg dm^-3 myoinositol, 10.0 µM BAP, 2.5 to 5.0 µM IBA and 0.5 mM spermidine.

The paper reports the in vitro cultivation of two commercial lines and 23 wild populations (with 10, 20 and 30 chromosomes) of Brachypodium distachyon. Callus induction was assayed on Murashige and Skoog medium containing 1 mg dm^-3 2,4-dichlorophenoxyacetic acid (2,4-D) with 30 g dm^-3 of sucrose (MSs) or maltose (MSm). No significant differences were seen between the two media with respect to callus induction. Calli were transferred to MSm medium without 2,4-D but containing 0.1 mg dm^-3 of 6-benzylaminopurine for plant regeneration. The plant regeneration response was very variable depending on the original induction medium, although no overall preference for one or the other medium was seen. The three main culture stages (callus induction, plant regeneration, and green plantlets formation) are probably differently controlled in the plants with different chromosome numbers. This supports the idea that the three cytotypes of Brachypodium cultured actually belong to different species.

Effects of four culture media on callus induction, regeneration and number of plants per unit culture were studied with mature seeds from five indica rice genotypes as explants. Based on the morphology, the calli were classified into four types as I to IV. Type I and type II are most suited to initiate suspension cultures or as target material for transformation. Number of plants regenerated per unit culture, formation of easily dissociating cell clusters and frequency of type I and type II calli were highest on NBKNB medium. Thus NBKNB medium is suitable for in vitro culture of even the hitherto recalcitrant indica genotypes.

Soil salinity leads to a reduction in plant growth, germination, relative water content, and production of wheat plants worldwide. Chitosan showed a positive effect on plant growth and development and improved plant stress tolerance. The current study aimed to examine the effect of different chitosan concentrations on the gamma-aminobutyric acid (GABA) shunt pathway in germinating seeds of wheat (Triticum durum L.) under salt stress (25 - 200 mM NaCl). We determined the seed germination pattern, seed moisture content, GABA shunt metabolites (GABA, glutamate, and alanine), oxidative damage in terms of malondialdehyde (MDA) accumulation, and the glutamate decarboxylase (GAD) mRNA transcription. Pre-treatment of wheat seeds with chitosan improved germination by enhancing germination percentage, seedling length, and seedling fresh and dry masses under salt stress. Data showed an increase in GABA shunt and their metabolites (alanine and glutamate), MDA content, and GAD mRNA transcription, and a decrease in germination percentage, seedling length, seedling fresh and dry masses for both untreated and chitosan-treated seeds under salt stress. Our results suggest that the elevation of GABA in chitosan-treated seeds was able to maintain metabolic stability under salt stress. The MDA content increased in chitosan-treated seeds as NaCl concentration increased, however, the increase was slightly lower than the MDA content in untreated seeds which confirmed that chitosan activates GAD mRNA expression that leads to activate GABA shunt to involve in the reduction of membrane damage and activation of reactive oxygen species scavenging systems under salt stress. Consequently, this study demonstrated that chitosan significantly enhanced the accumulation of GABA and amino acids metabolism to maintain the C:N balance and improved salt tolerance in wheat seeds during seed germination.

Callus induction was obtained on Murashige and Skogg agar medium with 45 μM 2,4-dichlorophenoxyacetic acid under dark at 25°C. Among the four explant types investigated, the best callus induction was obtained from two-week old fronds to which a surgical incision was applied in the basal (meristematic) region. This treatment resulted in 89.11% of fronds producing callus which continued to proliferate for another 24 months. To obtain plant regeneration pieces of calluses were transferred onto Murashige and Skoog agar medium containing 22 μM indole-3-acetic acid and 4.6 μM kinetin and maintained under 16-h photoperiod (irradiance of 30 μmol m^-2 s^-1) at 23°C. Green fronds formed on all callus pieces. The regenerated fronds were later transferred onto Wang medium where they formed roots. The regenerated Lemna minor L. plants obtained through indirect organogenesis did not differ morphologically from individuals forming the stock collection.

An efficient in vitro regeneration protocol was developed for medicinally important aromatic plant Anethum graveolens. Nodal segments were cultured onto Murashige and Skoog (MS) basal medium supplemented with different auxins and cytokinins singly as well as in combinations. The optimum callus induction (93.33 %) was obtained on medium fortified with 2.2 μM N⁶-benzyladenine (BA) and 0.21 μM α-naphthaleneacetic acid. The best shoot regeneration (85.7 %) with 12.86 shoots per explant was achieved in two weeks when callus was subcultured on MS medium amended with 2.2 μM BA and 1.85 μM kinetin. The average length of regenerated shoots varied from 3.15 to 4.8 cm. The rooting of regenerated shoots was nearly 100 % on 1/4 MS augmented with 4.9 μM indolebutyric acid with a maximum root length of 5.1 cm. Plantlets were successfully acclimatized with 60 % survival rate. During organogenesis, catalase and ascorbate peroxidase activity increased while superoxid dismutase activity decreased. Clonal fidelity of in vitro raised plants has been checked by random amplified polymorphic DNA using 10 selected decamer primers. It has been found that regenerated plants are true to type plants.

It is well known that phloem is essential for transporting proteins as long-distance signals. In this study, a proteomic approach was carried out to identify proteins accumulated in phloem exudates at the pre-floral induction (PFId) stage, at the floral induction (FId) stage, and at the floral initiation (FIn) stage. As a result, 45 phloem exudate proteins were detected. Six proteins were found at all three stages. Sixteen proteins were specific to the PFId stage, 14 proteins were specific to the FId and FIn stages. From the enriched gene ontology (GO)-terms related to transport, signaling, hormone, and development, 12 transport-related, 5 signaling-related, 10 hormone-related, and 9 development-related proteins were identified. It was found that arginine glycine glycine repeats nuclear RNA binding protein A (RGGA) was specific to the FId stage whereas heat shock protein 90-7, plasma membrane intrinsic protein 1-4, and the homolog protein encoded by At4g27190 were specific to the FIn stage. The relative abundances of sporulation protein F10D13.7 and ATP-dependent binding casette transporter G family member 37 were higher at the FId stage than at the PFId stage, and that of enolase 2 was higher at the FIn stage than at the PFId stage, suggesting that these phloem exudate proteins might act as long-distance signals involved in the transition from vegetative growth to reproductive growth in litchi. This study contributes to the comparative proteomic analysis of plant phloem sap, which will provide insights into proteins involved in floral induction as well as inter-organ communication during development in litchi.

The seasonal patterns of changes in the content of lipophilic antioxidants β-carotene (β-Car), zeaxanthin (Zx), α-tocopherol (α-Toc), plastoquinone (PQ)/plastoquinol (PQH₂) were studied in the assimilating shoots of evergreen shrub Ephedra monosperma J.G. Gmel ex C.A. Mey under natural conditions of Central Yakutia. The shortening of the photoperiod and the seasonal decrease in temperature induced a 1.4-fold increase in α-Toc content. The fall in the average daily temperature from 0.1 to -8.1°C in October led to a decrease in the content of β-Car as a result of the accumulation of rhodoxanthin (Rhd). In this period a sharp increase in the content of Zx retained overnight was also detected. In winter, elevated content of Zx and α-Toc persisted. During September, the content of PQH₂ increased by 2.5 times and PQ by 1.4 times (compared to July). The beginning of exposure to freezing average daily temperatures from -3 to -5°C led to the depletion of the total PQ pool by 18%. However, the content of PQ_tot in the winter months was 1.5 times higher than at the end of July. The results revealed different timing and temperature ranges of variation for individual antioxidants during the development of frost resistance in ephedra.

Interactions between indole-3-acetic acid (IAA), abscisic acid (ABA), and ethylene (ET) in the photoperiodic flower induction of a short-day (SD) plant Pharbitis nil were investigated. It was shown that both IAA and ABA applied just before and during the first half of the 16-h-long inductive night inhibited flower induction in P. nil. Ethylene is also thought to be a strong flowering inhibitor of SD plants but only when it is applied in the second half of the inductive night. The application of IAA just before the inductive night decreased the content of endogenous ABA in cotyledons only during the first half of the inductive night. Additionally, the application of 2-aminoethoxyvinylglycine (AVG) - an ethylene biosynthesis inhibitor - partially reversed the inhibitory effect of IAA and ABA on flowering. The results suggest that the mechanisms of P. nil flower inhibition by IAA and ABA might be independent. However, both the hormones influenced ethylene production which directly inhibited flowering. We also show that ABA applied on the cotyledons of P. nil seedlings just before the inductive night caused a clear increase in the expression of PnACS1 and PnACS2 genes (encoding enzymes involved in ethylene biosynthesis) from the first hours after its application. The transcripts of PnACO1 and PnACO3 genes were also increased but their maximal values were shifted in relation to the PnACS1 and PnACS2. The data presented here strongly support the idea that both IAA and ABA inhibit P. nil flowering through the modulation of ethylene biosynthesis.

A reproducible in vitro regeneration system for Nepalese kutki (Picrorhiza scrophulariiflora Pennell) was developed from in vitro leaf derived callus. Induction of more than seven shoot buds per explant was achieved on Woody plant medium (WPM) supplemented with 0.53 μM α-napthaleneacetic acid (NAA) and 0.23 μM kinetin (KIN). The shoots were elongated on WPM supplemented with 0.44 μM 6-benzylaminopurine (BAP) and rooted on WPM supplemented with 5.3 μM NAA within 2 weeks. The random amplified polymorphic DNA (RAPD) analysis indicated genetic uniformity of the micropropagated plants with its donor plants.

Interactions between three genotypes of a silver fir (Abies alba Mill.) embryogenic callus and Heterobasidion abietinum, H. parviporum, and H. annosum were examined in dual cultures. The aim of this study was to determine whether dual cultures can be used to evaluate the degree of fungal virulence at an embryogenic level, and whether different genotypes of a callus show different susceptibility. The dual cultures were performed on Schenk and Hildebrandt medium. Mycelial growth of H. parviporum and H. annosum was significantly stimulated in the presence of the callus but was not directional in nature. The embryogenic callus died between six and nine days after being colonized by H. parviporum suggesting that this was the most virulent species. By contrast, the callus remained healthy for up to 50 d after colonization with H. abietinum suggesting that this was the least virulent species. The callus of the A. alba genotype which originated in the mountain region of Poland remained healthy and alive for a significantly longer period than the other two genotypes in the dual cultures with all three Heterobasidion species even though overgrown by mycelium suggesting that the mountain genotype had the strongest defence response to Heterobasidion infection.

Plantlet regeneration through indirect somatic embryogenesis was attempted from rhizome derived callus of Cymbopogon winterianus Jowitt (cv. Jorlab2). Optimum callus was induced on Murashige and Skoog (MS) basal medium supplemented with 4 mg dm^-3 2,4-dichlorophenoxyacetic acid (2,4-D). Initially the callus was friable, shiny white and watery in nature. After subculturing on MS medium containing 2,4-D and kinetin (Kn), callus was transferred onto the MS medium supplemented with 2,4 -D, Kn and coconut water to induce somatic embryogenesis. Optimum somatic embryogenesis (78.33 %) was achieved on MS medium containing 3.0 mg dm^-3 2,4-D and 0.5 mg dm^-3 Kn. High frequency (65 %) plantlet conversion from embryos was achieved in MS medium supplemented with 2 mg dm^-3 N⁶-benzyladenine (BA), 0.5 mg dm^-3 Kn, 0.2 mg dm^-3 calcium pantothenate and 0.2 mg dm^-3 biotin.

A two-step protocol for improving the frequency of shoot regeneration from oilseed rape (Brassica napus L.) hypocotyl explants was established. The protocol consists of a pre-culture on callus induction medium (CIM) and a subsequent shoot regeneration on shoot induction medium (SIM). The SIM was Murashige and Skoog medium supplemented with different concentrations of 6-benzylaminopurine (BA; 2-5 mg dm^-3) and naphthaleneacetic acid (NAA; 0.05-0.15 mg dm^-3). Maximum frequency of shoot regeneration (13 %) was on the SIM medium containing 4 mg dm^-3 BA and 0.1 mg dm^-3 NAA, but it increased to 24.45 % when 20 μM silver thiosulphate (STS) was added. Strikingly, an extremely high frequency of shoot regeneration up to 96.67 % was reached by a two-step protocol when hypocotyl explants had been pre-cultured for 7 d on a CIM medium containing 1.5 mg dm^-3 2,4-dichlorophenoxyacetic acid. In addition, the shoot emergence was also 7 d earlier than that observed by use of the one-step protocol. The two-step protocol was also applied for regeneration of transgenic plants with cZR-3, a nematode resistance candidate gene. As a result, 43 plants were generated from 270 shoots and from these 6 plants proved to be transgenic.

The effect of heterosis on callus induction, callus mass and number of regenerated plants from mature embryo cultures of winter durum wheat (Triticum durum Desf.) hybrids was studied. A total of 14 F₁ hybrids and their parents were used for mature embryo culture. The statistical analysis of the results revealed that positive heterosis in callus mass was determined in only one F₁ hybrid and in number of regenerated plants in two F₁ hybrids. Plants regenerated in vitro were successfully established in soil. Hybrid genotypes may be used to obtain callus and regenerated plants with vigour comparable to their parents.

Leaves of Lotus japonicus infected by powdery mildew accumulate isoflavonoids, especially vestitol and sativan as protective compounds, whereas their flavonol-glycosides or phenylpropanoic acids content were rather decreased. For a better understanding of the induction of phytoalexin production in L. japonicus, the influence of important signalling molecules, namely methyl jasmonate and salicylic acid, was tested by leaf application, up to 15 d. No effects after spraying the leaves with 0.5 mM salicylic acid were observed regarding the plant isoflavonoid content, whereas methyl jasmonate showed moderate effect on vestitol accumulation. Chitosan application led to a strong increase in vestitol content, sativan and vestitol derivative also increased. In a similar way to the effect of powdery mildew infection, the content of ferulic acid and kaempferol glycosides decreased, except for one compound, identified as kaempferol-6-deoxyhexose, that is the result of acetylation of kaempferol glycosides. The application of chitosan also led to an approximately 2.5- to 3.5-fold increase in hydrogen peroxide content, indicating the involvement of H₂O₂ in the chitosan signalling pathway. The activity of key enzyme of the phenolic biosynthesis, phenylalanine amonia lyase (PAL), was stimulated as well as the expression of 3 PAL-isogenes (LjPAL4, LjPAL6, and LjPAL9), whereas LjPAL8 decreased. Moreover, the expression of two key enzymes of vestitol and sativan biosythesis: pterocarpan reductase and isoflavone synthase were also strongly induced. The data suggest that chitosan acts as an elicitor of the fungal attack in Lotus japonicus that do not lead to a general stimulation of the metabolism of phenols, but rather to a specific induction of isoflavonoid-phytoalexine production, especially for production of vestitol.

We have assayed different combinations of nutrient media and growth regulators to induce callus and plant regeneration from explants of root, shoot and leaf, complete seed, and isolated mature embryo of barley (Hordeum vulgare L. cv. Hassan). The best results were obtained with mature embryo in J25-8 medium supplemented with 2.0 mg dm^-3 2,4-dichlorophenoxyacetic acid where about 75 % developed friable calli. Some 80 - 85 % of these calli regenerated barley plants in the same J25-8 medium supplemented with 1.0 mg dm^-3 indole-3-butyric acid and 0.1 mg dm^-3 kinetin.

Ent-kaurene oxidase (EKO) catalyze three sequential oxidations in the early steps of gibberellin biosynthesis pathway. In this research, a cDNA sequence of InEKO1 gene in the model short-day plant Ipomoea nil was identified. Our studies revealed that inductive conditions for flowering caused an increase in the transcriptional activity of the examined gene in the cotyledons-the main organs for the perception of the photoperiodic stimulus. In contrast, in the second half of the 16 h long inductive night and after that, a decreased amount of InEKO1 mRNA in the apexes was detected. What is more, ethylene, the key inhibitor of flower induction in I. nil, elevated the InEKO1 expression exclusively in the cotyledons between 10 and 14 h of the inductive night.

Methods for induction of callus and cell suspension cultures have been developed for the medicinally important herb Centella asiatica (L.) Urban. Thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) analysis showed the presence of asiaticoside in the in vitro grown leaves, callus and cell suspension cultured cells.

During the normal developmental process, programmed gene expression is an essential phenomenon in all organisms. In eukaryotes, DNA methylation plays an important role in the regulation of gene expression. The extent of cytosine methylation polymorphism was evaluated in leaf tissues collected from the greenhouse grown plants and in in vitro-derived callus of three lowbush and one hybrid blueberry genotypes, using methylation-sensitive amplification polymorphism (MSAP) technique. Callus formation started from the leaf segments after 4 weeks of culture on a thidiazuron (TDZ) containing medium. Maximum callus formation (98 %) was observed in the hybrid blueberry at 1.0 mg dm^-3 TDZ. Although noticeable changes in cytosine methylation pattern were detected within the MSAP profiles of both leaf and callus tissues, methylation events were more polymorphic in calli than in leaf tissues. The number of methylated CCGG sites varied significantly within the genotypes ranging from 75 to 100 in leaf tissues and from 215 to 258 in callus tissues. Differences in the methylation pattern were observed not only in a tissue-specific manner but also within the genotype in a treatment specific manner. These results demonstrated the unique effect of TDZ and the tissue culture process on DNA methylation during callus development.

Most perennial and winter annual temperate grasses have a vernalization requirement (VR) for flowering, that is, they require a cold period before they can flower in response to long days. From a F1 mapping population of the outbreeding perennial forage grass Festuca pratensis Huds. (meadow fescue) previously used to map several quantitative trait loci (QTLs) for VR, we produced two F2 populations divergently selected for high or low VR. The two populations were characterised for flowering behaviour and gene expression of VRN1 as well as other MADS-box genes with a putative function in the induction of flowering. Expression of FpVRN1 and the VRN1-like genes FpMADS2 and FpMADS3 was associated with flowering but the response of gene expression to vernalization differed between genes and populations. The expression of the SVP-like genes FpMADS10 and FpMADS16 was not affected by vernalization and did not differ between the two F2 populations.