Explant Type Research Articles

Long-term organogenic callus cultivation of Ranunculus illyricus L.: a blueprint for sustainable ex situ conservation of the species in urban greenery

The growing trend of introducing wild plant species into urban environments necessitates the identification of novel species adapted to prevailing conditions. A promising reservoir of such species may be xerothermic communities where Ranunculus illyricus occurs. This study aimed to establish a micropropagation protocol for R. illyricus using indirect organogenesis. The protocol includes initiation of culture from various explants, callus proliferation, shoot regeneration, multiplication, and concurrent rooting. Callus appeared on most types of vegetative explants tested, but stolons were considered the best due to their good availability, high disinfection (85%), and robust callus production (maximum increase − 363.1%). The growth rate of the callus fresh matter (CFM) obtained from stolons was calculated. Greater CFM was obtained on the medium with the supplemented picloram 8.0 mg L− 1 with kinetin 5.0 mg L− 1 and in second part of experiment on medium with the addition of 2,4-D (2,4-dichlorophenoxyacetic acid) 2.0 mg L− 1 alone or picloram 6.0 mg L− 1 with kinetin 8.0 mg L− 1. Shoot organogenesis was observed on macronutrients B5 (Gamborg medium), micronutrients MS (Murashige and Skoog) medium with the addition of 2.0 mg L− 1 IBA (indole-3-butyric acid) and 4.0 mg L− 1 BAP (6-benzylaminopurine). To document the process of callus differentiation, microscopic preparations were prepared. Subsequently, the regenerated plants underwent acclimatisation and their growth in an ex situ collection was monitored over three growing seasons. In particular, in vitro-origin plants exhibited developmental patterns similar to those of their seed-origin counterparts. The incorporation of R. illyricus into urban landscapes not only increases aesthetic appeal, but also ensures the preservation of valuable genetic resources for this rare species, potentially contributing to effective ex situ conservation in the future. This marks the first scientific report on in vitro cultures of R. illyricus.

Organogenesis of Plant Tissues in Colchicine Allows Selecting in Field Trial Blueberry (Vaccinium spp. cv Duke) Clones with Commercial Potential

Plants’ genetic improvement continues to be crucial for modern agriculture, while biotechnology can offer efficient tools that enhance the selection and recommendation processes of elite clones. This work established a suitable methodology for the regeneration of blueberry (Vaccinium corymbsum) plants in cultures with colchicine. This could be considered a basis for producing populations for the selection of clones following a genetic improvement program assisted by biotechnology. The factors studied were: (a) explant type (leaf discs; nodal segments); (b) colchicine concentration (0, 0.5, 1, and 2 mg/L); and (c) time of exposure to colchicine (1, 2, 3, 5, and 30 days). The basal medium McCown’s Woody Plant (WP) supplemented with 2 mg/L 2iP and 1 mg/L BAP was used with the commercial genotype Duke as a model. A total of 1957 blueberry clones were produced in a medium with 1 mg/L colchicine, distributed at different exposure times. Flow cytometry analyses revealed the following patterns: single patterns for random samples of control plants (Duke donor) and some clones regenerated on colchicine; double patterns for chlorotic plants regenerated on colchicine. Triple and quadruple patterns were observed in callus tissues that did not regenerate plants on colchicine. Populations of plants regenerated in colchicine (6787) and control plants regenerated in in vitro culture without colchicine were adapted under greenhouse conditions. The variables evaluated at this stage were adaptability, height, diameter, number of leaves, incidence of diseases, flowering capacity, and agrobotanical traits. Selected clones demonstrating phenotypic variability (157 clones) were transplanted to field conditions. From the clonal field trial conducted under minimum tillage conditions, 38 clones were selected for improved traits related to the agricultural yield and nutritional quality of the fruits. Of these, six clones showed the highest agronomic performance and adaptability to adverse environmental conditions compared to the Duke donor genotype. It is recommended that these clones continue genotype × environment interaction trials at different locations.

Optimization of induction and hairy root culture establishment in two mullein species, Verbascum erianthum and Verbascum stachydiforme

The genus Verbascum, belonging to the family Scrophulariaceae, has a significant center of diversity in Iran. Two of its species, V. erianthum and V. stachydiforme, originate in the Iranian-Turanian region, but no studies have been conducted on the induction of their hairy roots. This genus is a valuable source of biologically active compounds such as iridoid glycosides and flavonoids. Hairy root culture is a suitable technique for the production and accumulation of secondary metabolites. Three different studies were conducted to optimize the induction and establishment of hairy roots. In the first experiment, the influence of explant type (leaf and hypocotyl), six infection methods, and co-culture time (48 and 72 h) on the efficiency of hairy root induction was investigated. The results showed that the highest hairy root induction (68.18%) was observed in the leaf explants inoculated by direct infection with three wounds within 72 h co-culture time. In the second experiment, the effect of four Agrobacterium rhizogenes strains (ATCC 15834, A4, A7, and A13) and leaf age (14, 21, and 28 days) on transformation efficiency and some morphological traits examined in both species were studied. The high transformation efficiency of hairy root (80.55%) was detected in the 21-day-old leaf explant of V. erianthum species that was inoculated with the A13 strain. The transformed hairy root colons were confirmed by PCR using rolB gene-specific primers. To optimize hairy root growth and avoid tissue browning, hairy roots were cultured in various media containing different antioxidants and improver agents (including ascorbic acid, citric acid, and NAA). The results showed that the highest fresh growth index (20.42) and the lowest tissue browning (9%) as well as total phenol (8.51 mg GA/g DW), and total flavonoid content (4.42 mg QUE/g DW) were obtained in medium B5 with 1.5 mg/l NAA.

Enhanced production of withaferin A from the hairy root culture of Withania somnifera via synergistic effect of Methyl jasmonate and β-cyclodextrin

Due to low amounts of withanolides produced in some plants and high demand for various applications, their biotechnological production is widely researched. The effects of two explant types (i.e., leaf and stem from the in vitro seedlings of three genotypes of Withania somnifera) and four Rhizobium strains (i.e., LBA 9402, A4, ATCC 15834, and C58C1) to improve hairy root formation efficiency was studied. Furthermore, the combined effects of β-cyclodextrin (β-CD) and methyl jasmonate (MeJA) on withaferin A production after 48 h exposure time was examined. Four hairy roots having the maximum percentage of induced roots and mean number of induced roots to analyze their growth kinetics and identified G3/ATCC/LEAF culture having the maximum specific growth rate (μ = 0.036 day−1) and growth index (GI = 9.18), and the shortest doubling time (Td = 18.82 day) were selected. After 48 h exposure of G3/ATCC/LEAF culture to different elicitation conditions, maximum amounts of withaferin A were produced in samples co-treated with 0.5 mM β-CD + 100 μM MeJA (9.57 mg/g DW) and 5.0 mM β-CD + 100 μM MeJA (17.45 mg/g DW). These outcomes represented a 6.8-fold and 12.5-fold increase, respectively, compared to the control. Similarly, combined β-CD/MeJA elicitation increased gene expression levels of HMGR, SQS, SMT-1, and SDS/CYP710A involved in withanolides biosynthetic pathway, of which just SMT-1 had significant correlation with withaferin A production. These results demonstrated the superiority of G1-leaf explant and ATCC 15834 for hairy root induction, and revealed synergistic effect of MeJA and β-CD on withaferin A production.

An Efficient Agrobacterium-Mediated Genetic Transformation System for Gene Editing in Strawberry (Fragaria × ananassa).

The octoploid-cultivated strawberry variety Benihope (Fragaria × ananassa Duch cv. Benihope) is an important commercial plant. It is highly susceptible to different diseases, which ultimately leads to a reduction in yield. Gene-editing methods, such as CRISPR/Cas9, demonstrate potential for improving disease resistance in the strawberry cv. Benihope. Establishing a plant regeneration system suitable for CRISPR/Cas9 gene editing is crucial for obtaining transgenic plants on a large scale. This research established a callus induction and plant regeneration system for Agrobacterium-mediated CRISPR/Cas9 gene editing in strawberry cv. Benihope by evaluating multiple types of explants and various plant growth regulators throughout the entire tissue culture process. The results showed that the efficiency of callus induction is strongly influenced by the type of explant and is highly sensitive to the combination of plant growth regulators. Among the different plant growth regulators employed, thidiazuron (TDZ), in combination with 2,4-dichlorophenoxyacetic acid (2,4-D), effectively induced callus formation and plant regeneration from explants derived from nutrient tissues such as runner tips and crowns. In addition, the regeneration experiment demonstrated that the addition of polyvinylpyrrolidone (PVPP) to the shoot regeneration medium could inhibit tissue browning. The gene-edited plants in which some or all of the Fvb7-1, Fvb7-2, Fvb7-3, and Fvb7-4 genes in the MLO (Mildew resistance Locus O) gene family were knocked out by CRISPR/Cas9 system were obtained by applying the plant regeneration system developed in this study.

Optimization of an Efficient Direct Shoot Induction System and Histological Evaluation of Shoot Formation in Cucurbita maxima Duch

Improving plant regeneration ability and shortening regeneration time can promote the development of genetic transformation breeding technology for horticultural crops. We optimized several culture conditions, including explant type, mother plant genotype, and medium, to improve shoot formation in winter squash (Cucurbita maxima Duch.). Histological analysis of the occurrence of shoots was also carried out. The results indicate that cotyledon was the most suitable explant for inducing the shoot regeneration of winter squash. We found that ‘Jin-li’ had a shorter shoot induction time and a higher average number of shoots. The highest induction rate of 95.23% among the five lines. The average shoot induction rate of five lines was the highest (84.85%) on Murashige and Skoog (MS) medium supplemented with 2.0 mg/L 6-benzylaminopurine (6-BA) and 0.2 mg/L indole-3-acetic acid (IAA). We also found that there was an interaction between genotypes and induction media, and their interaction had a greater impact on the shoot induction rate than individual effects. Histological observation revealed that the induced shoots of winter squash cotyledons originated from subepidermal cells. We also found that the optimal medium for de novo root regeneration was 1/2 MS. We acclimatized and cultivated regenerated plants and harvested their fruits, which maintained the characteristics of mother plants. These findings lay an important foundation for further research on direct shoot regeneration and accelerate its application in winter squash genetic transformation.

In Vitro Propagation Journey of Ornamental Gladiolus (Gladiolus Species): A Systematic Review Analysis Based on More Than 50 Years Research

Traditional gladiolus propagation methods are now supplemented with in vitro propagation to meet the demands of modern floriculture in terms of quick production of disease-free, quality planting material. Due to virus infections, vegetative propagation in gladiolus in the field is slow, and is a serious concern in the propagation of gladiolus. In vitro propagation provides an enormous increase in propagation rate and the ability to produce disease-free plant material. Numerous elements, including cultivars, explant type, size of explants, position of explants on medium, plant growth regulators and certain additives, incubation conditions, and sub-culturing time, all have a significant impact on in vitro clonal propagation of gladiolus plants as well as the development of in vitro cormel efficiency. There are certain obstacles and challenges that arise in the in vitro development of plants and the cormels of gladiolus. However, numerous studies and review reports on gladiolus for in vitro propagation have been reported, but very little is known about the factors influencing gladiolus’ in vitro effectiveness. In the present review, we focused on and analyzed research data accumulated over 50 years on diverse strategies for in vitro propagation such as direct, indirect organogenesis, and somatic embryogenesis, as well as various factors such as physical, nutritional, and hormonal influences on in vitro propagation, in vitro cormel formation efficiency, difficulties that arise, and new insights into in vitro development in gladiolus from the available literature worldwide. Future possibilities for further improvement in the in vitro propagation of ornamental gladiolus are also discussed. The current review provides insight into a comprehensive protocol for gladiolus in vitro propagation and emphasizes the importance of continuously advancing tissue culture techniques and factors influencing the in vitro efficiency towards improving in vitro plantlets and cormels in gladiolus (Gladiolus spp.).

Echinacea purpurea L. Kallus Kültürlerinde Karbon Kaynağı, Nitrojen ve PEG'in Kafeik Asit Türevlerinin Üretimine Etkisi

This study aimed to determine the effects of polyethylene glycol (PEG) as an abiotic elicitor and nutritional factors (different ammonium/nitrate ratios, carbon source and amount) in the culture medium on the production of Caffeic Acid Derivatives (CADs) in callus cultures of Echinacea purpurea L. Petiole and root explants were cultured on MS medium modified in terms of different types (sucrose and maltose) and amounts (sucrose 15, 45, 60 g l-1, and maltose 15, 30, 45, 60 g l-1) of carbon source, different concentrations (5, 10, 15 g l-1) of PEG and ammonium nitrate ratios (0:35, 5:25, 15:15, 35:0 mM). The amounts of CADs in the callus obtained at the end of the 10-week culture period were analysed. In both explant types, the highest amount of CADs were obtained from the medium containing 15 g l-1 sucrose and 15 or 30 g l-1 maltose applications, while the highest amount of CADs was obtained in the medium containing 0:35 mM ammonium/nitrate in nitrogen applications. While the highest amount of CADs in root explant was obtained from the medium containing 10 g l-1 PEG applications, CADs content could not be obtained in petiole explant. As a result, the highest amounts of caftaric, chlorogenic, caffeic, and chicoric acids (respectively, 9.38, 0.71, 0.29, and 34.77 mg g-1) were determined at callus obtained from root explant cultured on MS medium containing 30 g l-1 sucrose and 0:35 mM ammonium/nitrate. In conclusion, optimization of culture conditions and different elicitor applications were made to increase secondary metabolite content in E. purpurea L. under in vitro conditions and the results obtained were presented comparatively.

Machine learning-mediated Passiflora caerulea callogenesis optimization.

Callogenesis is one of the most powerful biotechnological approaches for in vitro secondary metabolite production and indirect organogenesis in Passiflora caerulea. Comprehensive knowledge of callogenesis and optimized protocol can be obtained by the application of a combination of machine learning (ML) and optimization algorithms. In the present investigation, the callogenesis responses (i.e., callogenesis rate and callus fresh weight) of P. caerulea were predicted based on different types and concentrations of plant growth regulators (PGRs) (i.e., 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzylaminopurine (BAP), 1-naphthaleneacetic acid (NAA), and indole-3-Butyric Acid (IBA)) as well as explant types (i.e., leaf, node, and internode) using multilayer perceptron (MLP). Moreover, the developed models were integrated into the genetic algorithm (GA) to optimize the concentration of PGRs and explant types for maximizing callogenesis responses. Furthermore, sensitivity analysis was conducted to assess the importance of each input variable on the callogenesis responses. The results showed that MLP had high predictive accuracy (R2 > 0.81) in both training and testing sets for modeling all studied parameters. Based on the results of the optimization process, the highest callogenesis rate (100%) would be obtained from the leaf explant cultured in the medium supplemented with 0.52 mg/L IBA plus 0.43 mg/L NAA plus 1.4 mg/L 2,4-D plus 0.2 mg/L BAP. The results of the sensitivity analysis showed the explant-dependent impact of the exogenous application of PGRs on callogenesis. Generally, the results showed that a combination of MLP and GA can display a forward-thinking aid to optimize and predict in vitro culture systems and consequentially cope with several challenges faced currently in Passiflora tissue culture.

Development and Optimization of a Rapid In Vitro Micropropagation System for the Perennial Vegetable Night Lily, Hemerocallis citrina Baroni

The perennial herbaceous night lily, Hemerocallis citrina Baroni, is an important vegetable crop with an increasing production and consumption in China. The long lifecycle and slow growth of the night lily are becoming bottlenecks for the large-scale production of elite lines and various genetic and breeding studies. There is a lack of a protocol for rapid and efficient micropropagation for this crop. Here, we reported the systematic investigation and optimization of in vitro plant regeneration through tissue-culture-based organogenesis in the night lily variety ‘Datong Huanghua’. We evaluated various factors affecting the efficiency of callus induction and subculture, shoot regeneration, rooting and plantlet establishment, including explant type and age, inoculation methods, basal culture media and the type and concentration of plant growth regulator (phytohormones) in various growth media. We developed an optimized protocol, as follows. The highest efficiency of callus induction was observed on Murashige and Skoog (MS) medium supplied with 22.7 µM TDZ (thidiazuron) using the young scape (flower stem or stalk) as the explant, which was cut longitudinally in half to produce a segment approximately 0.5 cm in length. Callus subculture and proliferation were more efficient on MS medium containing 9.0 µM 2,4-D (2,4-dichlorophenoxyacetic acid) under light culture conditions. Shoot regeneration showed the highest efficiency on MS medium supplemented with 8.9 µM 6-BA (6-benzylaminopurine) + 5.4 µM NAA (α-naphthaleneacetic acid), while the best rooting medium was MS medium containing 2.7 µM NAA. After transplanting, the transplanted regenerated seedlings showed the highest survival rate (96%) on a substrate mixture with a 2:1:1 ratio of peat/perlite/vermiculite. A protocol and flowchart for the rapid in vitro micropropagation of night lily plants is proposed that will facilitate various genetic, genomic and breeding studies on this crop.

Morphogenesis Response of Leaf and Petiole Explant of Rootstock Apple (Malus sylvestris Mill.) to Auxin and Cytokinin

Rootstock apple is a type of plant used in grafting propagation. Research related to the response of morphogenesis in this plant has not been widely carried out. The aim of this research was to study the effect of auxin and cytokinin ratio on the morphogenesis response of leaf and petioles explants of rootstock apple and to study variations in the ratio of auxin and cytokinin with the best response. The study used a 2-factorial Completely Randomized Design (CRD) with variations in the types of explants and combinations of plants growth regulators (PGR) in the culture medium. There were 12 treatments with five repetitions. Explants in the form of young apple leaf (third leaf) and petiole rootstock that had been sterilized and cut with a size of 5x5 mm for the leaf and 5 mm long for the petiole were inoculated into MS medium with cytokinin in the form of BAP (1 ppm) combined with auxin, IBA (0, 0.1, 0.2, 0.3, 0.4, 0.5 ppm). Each culture bottle contains five explants. The results showed that the average leaf explant was capable of producing 34% callus. Meanwhile, petiole explants produced 4% callus. Callus began to form on leaf and petiole explants on the 26th and 42nd days after inoculation respectively. Most calluses have a compact texture with variations in green, yellowish white, and brownish green. The PGR combination with the highest percentage of callus formation in leaf explants was found in the BAP 1 ppm + IBA 0.4 ppm treatment, while the petiole was in the BAP 1 ppm + IBA 0.3 ppm treatment

Tobacco Plant: A Novel and Promising Heterologous Bioreactor for the Production of Recombinant Bovine Chymosin.

The natural source of chymosin, a key enzyme in the dairy industry, is insufficient for rapidly growing cheese industries. Large-scale production of recombinant proteins in heterologous hosts provides an efficient alternative solution. Here, the codon-optimized synthetic prochymosin gene, which has a CAI index of 0.926, was subcloned from a cloning vector (pUC57-bCYM) into the pBI121 vector, resulting in the construct named pBI121-bCYM. CAI ranges from 0 to 1 and higher CAI improves gene expression in heterologous hosts. The overexpression of the prochymosin gene was under the control of constitutive CaMV 35S promoter and NOS terminator and was transferred into the tobacco via A. tumefaciens strain LBA4404. Explant type, regeneration method, inoculation temperature, cell density (OD600) of Agrobacterium for inoculation, and acetosyringone concentration were leaf explants, direct somatic embryogenesis, 19 °C, 0.1, and 100 µM, respectively. The successful integration and expression of the prochymosin gene, along with the bioactivity of recombinant chymosin, were confirmed by PCR, RT-PCR, and milk coagulation assay, respectively. Overall, this study reports the first successful overexpression of the codon-optimized prochymosin form of the bovine chymosin enzyme in the tobacco via indirect transformation. Production of recombinant bovine chymosin in plants can be an easy-to-scale-up, safe, and inexpensive platform.

In vitro Propagation of Ocimum Sanctum Linn by Using Growth Hormone Shoot Induction

The majority of Indian homes include tulsi, often known as holy basil (Ocimum sanctum L). Although it is regarded as a spiritual plant, physiologically speaking, it is one of the most readily available antibiotics. Tulsi is the most significant herb in Ayurveda, and current research is confirming its health advantages. It is also one of the plants that are utilised widely in Ayurvedic treatments.It relieves physical, physiological, metabolic, and psychological stress due to its special mix of pharmacological activity. A substrate for the quick development and multiplication of commercially significant plants is provided by plant tissue culture. Determining the optimal explants type and medium conditions for large-scale in vitro Tulsi shoot induction is the aim of the current effort. In the current investigation, the nodal segment and shoot tips were employed as explants. The nodal segment reacted well with a frequency rate of about 90% on all MS media utilized in the current investigation, including media with BAP and media with different combinations of BAP and IAA. It was seen that one or more shoots were emerging from the explants' nodal area after 10 days of culture. In this work, we examined the effects of various PGR combinations and dosages on the in vitro micro propagation of Tulsi, a fragrant and therapeutic plant (Ocmium sanctum L.). Three distinct PGRs were utilized, namely 6-benzylaminopurine (BAP), naphthalene acetic acid (NAA), and indole-3-acetic acid (IAA). The optimal medium for inducing and multiplying shoots was found to be Murashige and Skoog (MS) medium supplemented with 0.25 mg/l BAP and 0.1 mg/l NAA. The MS medium exhibits average shoot formation with 0.025 mg/l IAA and 0.1 mg/l BAP. Our results demonstrate that Tulsi may be successfully micro-propagated in vitro with the appropriate PGR.

Callusogenesis and Organogenesis of Thymus Cultivars In Vitro

The article is devoted to the in vitro cultivation of three Russian cultivars of two Thymus species (Th. vulgaris and Th. serpyllum) in order to study the possibility of induction of callusogenesis and organogenesis. The developed technology opens up prospects for the induction of somaclonal variability, obtaining the source material for breeding, as well as regeneration of thyme plants after genetic transformation. For the study, we used nutrient media based on the mineral composition of the Murashige and Skoog medium with the addition of auxin (indole-3-butyric acid, indole-3-acetic acid) and cytokinin (6-benzyladenine, kinetin) substances. For the induction of callusogenesis, the use of stem explants on nutrient media supplemented with 2,4-dichlorphenoxyacetic acid (1 or 2 mg/L) can be recommended. The efficiency of shoot organogenesis also depends on the type of explant, and its rather low values in most experimental variants are apparently due to the accumulation of endogenous auxins and phenolic compounds. Thus, a cultivar-specific morphogenetic response to the use of various phytohormones and growth regulators was established, as well as the influence of the explant type on the efficiency of morphogenic processes.

Micropropagation of Dipcadi montanum (Dalz.) Baker (Asparagaceae): a rare scapigerous herb.

Dipcadi montanum (Dalz.) Baker (Asparagaceae) is a rare scapigerous herb endemic to the Western Ghats, a global biodiversity hotspot running parallel to the western coast of India. This study reports the development of a reproducible protocol for mass propagation of this underutilized geophyte using bulb scale and immature leaf base explants. Miniature bulblets were successfully induced from both types of explants after 4 and 8 weeks of culture on full-strength semisolid MS basal medium fortified with 3% sucrose and varying levels of BAP (4.4-17.7 μM) and TDZ (4.5-18.1 μM). The addition of 2.7 μM NAA further enhanced the rate of microbulb induction. Rooting of the 8-week-old bulblets, obtained from both explants, was achieved with more than 90% efficiency on liquid as well as agar-gelled half-strength MS basal medium fortified with varying levels of IBA (2.46-9.84 μM) and NAA (2.68-10.74 μM), with or without 2.32 μM Kinetin. More than 95% of the rooted plants survived the initial acclimatization process under controlled ex-vitro conditions, and a survival rate of over 80% was recorded after 4 weeks of transfer to greenhouse conditions. After a brief dormancy, the regenerants resumed growth in the postmonsoon season and exhibited morphological resemblance to the donor plant. Comparative cytological analysis between the donor and 15 randomly selected regenerants revealed a stable somatic count of 2n = 20.

Callus Induction and Establishing Cell Suspension Culture of Agastache J.Clayton Ex Gronov

The aim of this study was to initiate calli formation and cell suspension cultures from some species of Agastache genus. These plants could be useful for a production of bioactive secondary metabolites in vitro. For the initiation of callogenesis, two explant types were tested: leaf and stem explants from 40–60 days old in vitro seedlings. Percentage of callus formation was used as criterion to evaluate the efficiency of callus induction. Leaf- and stem-derived friable calli of A. foeniculum and A. urticifolia cultivated on MS medium supplemented with 0.5 mg/L 2.4-dichlorophenoxyacetic acid and 0.1 mg/L kinetin were selected for the cell suspension cultures establishing. The cell suspension cultures of A. foeniculum characterized by growth indexes of 1.08 and 8.57 for MS and B5 media respectively. For A. urticifolia suspension cultures growth indexes were 3.01 for MS medium and 1.29 for B5 medium. The period of culturing was 28 days. Viability of cell suspension cultures varied 50–100 during the period of culturing. According to the growth characteristics for establishing A. foeniculum suspension culture is better to use MS medium, and for A. urticifolia – B5.

Proliferation of Axillary Shoots of Chestnut in Temporary Immersion Systems.

Shoots from European chestnut and hybrids of European and Asian chestnuts can be efficiently proliferated in liquid medium by temporary immersion (TIS) using RITA® and Plantform™ bioreactors. The main challenges of applying TIS to these species were the lack of growth and hyperhydricity; problem solutions included the manipulation of the hormone concentration, the explant type, immersion frequency, and a support for maintaining shoots in a vertical position. After protocol optimization, explants cultured by TIS produced more rootable shoots than explants growing in semisolid medium, enabling increased number of rooted and acclimatable shoots. In this chapter, we will describe the protocols for proliferating chestnut by TIS in RITA® and Plantform™ bioreactors, together with tips for avoiding the main pitfalls of the technique. The strategies applied to chestnut can be useful for culturing other woody plants in bioreactors.

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Establishment of an Effective in vitro Regeneration System for Sugar Beet (Beta vulgaris L.)

Plant regeneration and dedifferentiation are known to be difficult for sugar beet (Beta vulgaris L.) due to its recalcitrant nature. This leads to low transformation efficiency and thus successful application of plant molecular techniques is limited in sugar beet for its genetic enhancement. A prolific regeneration method has been established by modulating several plant growth regulators on the in vitro regeneration of Beta vulgaris var. V6 KWS Serenada. Several types of explants excised from young seedlings of this variety were used for both direct and indirect regeneration of shoots. The highest response towards direct shoot formation and callus induction were obtained from cotyledonary nodes and hypocotyls, respectively. Explants were cultured on MS medium supplemented with different concentrations of BAP, GA3 and 2, 4 -D for callus induction as well as formation of shoots. Hypocotyls responded well for callus induction on MS medium containing 0.1 mg/l BAP + 2.0 mg/l 2, 4-D, while cotyledonary nodes exhibited the highest responses towards shoot formation on MS medium containing 1.0 mg/l BAP and 1.5 mg/l GA3. MS medium containing 2.0 mg/l IBA produced the highest number of roots per shoot. The in vitro raised rooted plantlets were successfully transferred to soil for acclimatization. Plant Tissue Cult. & Biotech. 33(2): 181-188, 2023 (December)

Comprehensive Analysis of Highbush Blueberry Plants Propagated In Vitro and Conventionally.

In vitro culture allows the production of numerous plants with both desirable and undesirable traits. To investigate the impact of the propagation method on highbush blueberry plants, an analysis was performed on four groups of differentially propagated plants: in vitro with axillary (TC-Ax) or adventitious shoots (TC-Ad), conventionally (SC) and using a mixed method (TC/SC). The analysis included plant features (shoot length and branching, chlorophyll and fluorescence and DNA methylation) and fruit properties (antioxidant compounds). The data obtained indicated significant differences between plants propagated conventionally and in vitro, as well as variations among plants derived from in vitro cultures with different types of explants. SC plants generally exhibited the lowest values of morphological and physiological parameters but produced fruits richest in antioxidant compounds. TC/SC plants were dominant in length, branching and fluorescence. Conversely, TC-Ax plants produced fruits with the lowest levels of antioxidant compounds. The methylation-sensitive amplified polymorphism (MSAP) technique was employed to detect molecular differences. TC-Ad plants showed the highest methylation level, whereas SC plants had the lowest. The overall methylation level varied among differentially propagated plants. It can be speculated that the differences among the analysed plants may be attributed to variations in DNA methylation.