Concentrations Of Thidiazuron Research Articles

Influences of Thidiazuron Concentrations and Exposure Duration on Axillary Shoot Proliferation of Faba Bean [Vicia faba (L.)] Genotypes

Background: Faba bean is one of the most important feed grain legume crops in the world. However, this crop is a recalcitrant species for in vitro regeneration. Methods: In this study, in vitro shoot proliferation of ten faba bean genotypes, that are classified according to their seed size into small, medium and large, were investigated in response to different light regimes and thidiazuron (TDZ) treatments. Result: Incubation under different light regimes, explant type as well as genotypes influenced seedling growth and axillary shoot multiplication of faba bean. The explants with single cotyledon and incubated under continuous darkness for 6 days resulted higher number of shoot than nodal explants. Medium size seeded genotype (ILB4347) produced the highest shoot and root length and shoot proliferation as compared with large (Luz) or small (Triple White) seeded genotypes. The effects of TDZ concentrations (2-10 mM/L) and exposure duration (10 and 20 days) on axillary shoot multiplication were investigated for the ten faba bean using cotyledonary explants. The overall mean of TDZ time exposure showed that treatment of 20 days exposure had higher number of shoot compare to 10 days exposure treatment with 3.77 shoot per explant. On the basis of genotype used, the three most responsive genotype in tissue culture were medium sized seeds genotypes (ILB4347, Hassawi 2 and Misr 3 with 4.76, 4.40 and 4.15 shoot per explant, respectively). ILB4347 were the most responsive genotype in tissue culture. Treatment of 8 mM TDZ after 20 days followed by 2 weeks on MS medium without PGRs generated the highest proliferation with 6.63 shoot per explant. These findings contribute towards effective in vitro propagation of faba bean.

In vitro production of Phalaenopsis orchids

Phalaenopsis – known as moth orchids – are the most popular orchids cultivated indoors as decorative house plants. This makes propagation and cultivation of Phalaenopsis important for commercial growers. Enhancements to the micropropagation of Phalaenopsis would have pronounced economic benefits through reduced losses and wastage. We examined the effects of several nutrient media and specific plant growth regulators (PGRs) belonging to the gibberellic acid and cytokinin groups on the in vitro germination of Phalaenopsis seeds, utilizing a single-group pretest-posttest model. The effects of several nutrient media such as: Knudson C (KCM), Lindemann (LM), Orchimax (-OM), Orchimax + activated charcoal (+OM), Murashige & Skoog (MS), as well as various PGRs such as 6-Benzylaminopurine (6BA), 6-Furfurylaminopurine (KIN), Adenin hemisulfate (AHS), Thidiazuron (TDZ), 2-Isopentenyl adenine (2iP), and Gibberellic acid (GA3), on the process of germination were also investigated. The explants obtained from the germinating seedlings were subjected to direct organogenesis, and the optimal PGR and tissue fragments were determined. The +OM medium facilitated the shortest germination period (in days). An inverse relationship between the concentration of TDZ and the percentage of germination in the context of the employed PGRs was observed. Apart from TDZ, the remaining PGRs exhibited a positive correlation with concentration. However, no significant difference in germination was observed in comparison to the control. The findings of direct organogenesis investigations revealed that the medium that exhibited the highest productivity was enriched with 5.0 ppm of 6BA. The media containing TDZ exhibited a reduced level of efficiency. Particularly, the group treated with 1.0 ppm of TDZ exhibited reduced efficacy compared to the control group. All concentrations of cytokinin in root elongation stage exhibited a favorable impact in comparison to the control. The variance between these PGRs was not statistically significant.

Combination of thidiazuron and basal media type on optimizing in vitro growth of Grammatophyllum stapeliiflorum orchids

Grammatophyllum stapeliiflorum, recognized as the melancholic orchid, represents one of the elusive orchid species facing the brink of extinction, thus exacerbating its rarity. Tissue culture technology becomes imperative for the propagation of this orchid species. This study aims to scrutinize the influence of different concentrations of the synthetic growth regulator thidiazuron (TDZ) on distinct basal media types, Murashige Skoog (MS) and Vacin and Went (VW), on the in vitro growth of G. stapeliiflorum orchids. Employing a complete randomized factorial design, the study entails two research factors: TDZ concentration with 4 treatment levels, A0 = 0 mg/L, A1 = 0.25 mg/L, A2 = 0.50 mg/L, and A3 = 0.75 mg/L, and the second factor being the media type, B1 = ½ MS and B2 = ½ VW. Findings indicate significant effects of MS and VW media on the percentage of viable explants, browning, globular forms, and shoots. Interaction between TDZ concentration and media type didn't yield significant effects on each experimental parameter. Treatment with 0.25 and 0.50 mg/L TDZ on ½ VW media demonstrated optimal results for explant growth percentage, browning, and explants in the globular phase, at 97.33%, 2.67%, and 2.67%, respectively. Moreover, treatment with 0.75 mg/L TDZ on ½ VW media resulted in the highest percentage of explant coloration. The combination of TDZ and media has shown the potential to enhance the production efficiency and conservation of this rare orchid species through tissue culture technology.

Enhancing Clitoria ternatea Propagation Through Indirect Somatic Embryogenesis: Optimal Plant Growth Regulators Type and Concentration for Efficient in Vitro Plant Production

The propagation of Clitoria ternatea poses several challenges, including low germination rates, vulnerability of young seedlings in natural environment, and a slow flowering rate. This study aims to identify the best Plant Growth Regulators and their optimal concentration to stimulate indirect somatic embryogenesis from C. Ternatea leaves explants. The samples were cultured on Murashige and Skoog (MS) medium supplemented with various concentration of thidiazuron (TDZ; 1, 2, 3, 4, and 5 mgL-1 ) and a combination of 2,4-dichloro phenoxy acetic acid (2,4-D; 1, 2, and 3 mgL-1 ) and 0.1 mgL-1 kinetin. The findings revealed that all treatments resulted in the formation of both embryogenic and non embryogenic calluses. The addition of TDZ to the MS medium led to a greater percentage of embryogenic callus formation compared to using the combination of kinetin and 2,4-D. The highest percentage of 84.37% was achieved when samples were cultured on MS medium with 3 mgL-1 TDZ. The most effective induction period of 21 days was observed with the medium supplemented with 3 mgL-1 TDZ, followed by 4 and 5 mgL-1 TDZ, which took 23 days. The research findings suggest that a concentration of 3 mgL-1 TDZ is the optimal PGR type and dosage for achieving rapid and efficient in vitro plant production compared to various concentrations of 2,4-D in combination with kinetin.

The Effect of Cow Stage Fertilizer Dosage and Thidiazuron Concentration on The Growth of Porang (Amorphophallus Oncophyllus) Seeds

This research aims to determine the effect of the dose of cow manure and the concentration of the growth regulator Thidiazuron on the growth of porang plant seeds. The design used was a factorial randomized block design consisting of 2 factors. Factor I, dose of cow manure, and factor II concentration of PGR Thidiazuron. This research concludes: (1) The dose of cow manure has a significant effect on the day shoots emerge, plant height at 75 DAP, stem diameter at 45, 60, and 75 DAP, and number of leaves at 75 DAP, and has a very significant effect on height. plants at 45 and 60 DAP and wet stover weight at 75 DAP, had no significant effect on the vigor index on the 7th day after DAP. (2) Thidiazuron PGR concentration had a very significant effect on the day of shoot emergence, vigor index on the 7th day of DAP, and plant height at 45 and 60 DAP, had no significant effect on plant height at 75 DAP, stem diameter at 45, 60 and 75 DAP, number of leaves 75 DAP and wet stover weight 75 DAP. (3) The interaction between the treatment dose of cow manure and the PGR concentration of Thidiazuron had a significant effect on the number of leaves. The best results from this research were in the treatment with a dose of cow manure of 15 g/polybag and a Thididazuron PGR concentration of 3 mg/l of water.

In Vitro Regeneration of Chrysanthemum from Ovaries and Ovules Treated with Thermal and Chemical Stimuli: Morphogenic and Cytogenetic Effects

Chrysanthemum (Chrysanthemum × morifolium (Ramat.) Hemsl.) holds a prominent position in the market of ornamental plants. To further advance chrysanthemum breeding efforts, the development of haploids may be useful. Therefore, the effect of various chemical and thermal treatments on regeneration efficiency and ploidy level in chrysanthemum was studied. Ovaries and ovules of three chrysanthemum cultivars, i.e., ‘Brasil,’ ‘Capitola,’ and ‘Jewel Time Yellow,’ were cultured either on a medium with 1 mg·L−1 2,4-dichlorophenoxyacetic acid (2,4-D) and different concentrations (0.5–1.5 mg·L−1) of thidiazuron (TDZ) or subjected to thermal shock (pretreatment temperature of 4 °C or 32 °C) and cultured on a medium with 1 mg·L−1 2,4-D and 1 mg·L−1 6-benzylaminopurine (BAP). It was found that ovaries had a greater organogenic potential (both in terms of callogenesis and shoot formation) than ovules. Microscopic analyses revealed that shoots mainly developed via indirect somatic embryogenesis from a callus developed from the ovary wall. The highest number of shoots was produced in cooled (at 4 °C) ovaries of chrysanthemum ‘Brasil’ and in ‘Jewel Time Yellow’ ovaries cultured on a medium with 1.0–1.5 mg·L−1 TDZ. The latter cultivar also had the highest potential to produce plants with an altered ploidy level (doubled and halved the number of chromosomes). This study demonstrates that manipulating factors such as temperature and thidiazuron concentration can enhance regeneration efficiency and induce altered ploidy levels in selected cultivars, offering valuable insights for chrysanthemum breeding programs.

Synergistic effects of Vermi-Co extracts mixture with cytokinins on shoot regeneration of Lindernia minima (Benth.) Mukerjee—An endangered medicinal plant

The present investigation illustrates the synergistic growth effect of Vermi-Co (Vermicast and Coconut-water) extracts mixture with cytokinins on in vitro shoot regeneration of Lindernia minima. Due to less distribution threat status, this plant species has been considered as an endangered plant by IUCN (International Union for Conservation of Nature). Hence, it is necessary to conserve and to develop an efficient plant regeneration protocol for the rapid multiplication of L. minima. The nodal explants were inoculated on (¼) quarter-strength MS (Murashige and Skoog) medium supplemented with different concentrations (0.5 – 2.5 mg L-1) of BA (6-Benzyladenine), KIN (Kinetin) and TDZ (Thidiazuron) combined with various percentages of (5%, 15%, and 25%) Vermi-Co extracts mixture. Among the concentrations tested, 1.5 mg L-1 TDZ with 15% of Vermi-Co extracts mixture produced the maximum percentage of response with the highest (19.5 ± 0.20 shoots/explant) number of shoots and (10.2 ± 0.14 cm) shoot length. Then, the well-elongated shoots were transferred to rooting medium (quarter-strength MS) supplemented with 0.5 – 3.0 mg L-1 of IBA (Indole -3- butyric acid) and IAA (Indole -3- acetic acid). The highest percentage of response (98%) and maximum number of roots were observed in 1.5 mg L-1 IBA with 11.5 ± 0.29 roots per shoot and 6.6 ± 0.28 cm root length. Finally, well-developed plants were successfully transferred to the natural field condition with a survival rate of 97%. The present research revealed that, the combined growth stimulating effect of Vermi-Co extracts mixture combined with cytokinins increased the number of shoots and shoot length on shoot regeneration of L. minima from nodal explants. Our findings will be very useful for regeneration of Lindernia minima through plant tissue culture.

Optimizing Different Medium Component Concentration and Temperature Stress Pretreatment for Gynogenesis Induction in Unpollinated Ovule Culture of Sugar Beet (Beta vulgaris L.)

The great economic importance of sugar beet determines the ongoing biotechnological studies conducted worldwide to improve the technology of obtaining doubled haploids (DHs) using the method of unpollinated ovule culture in vitro. To improve the induction of gynogenesis, we tested the effect of thidiazuron (TDZ), temperature bud pretreatment, different concentrations of sucrose, and culturing on liquid or solid medium. Three genotypes were tested in this study. The use of TDZ at a concentration of 0.4 mg/L in solid IMB (induction medium for Beta vulgaris) induction nutrient medium with 3 g/L phytagel, 50 g/L sucrose, 200 mg/L ampicillin and cultivation at 28◦C in the dark produced up to 16.7% induced ovules. The liquid nutrient medium of the same composition induced up to 8% ovules. Increasing TDZ concentration to 0.8 mg/L resulted in reduction or total inhibition of gynogenesis, depending on the genotype. Reducing the sucrose concentration to 20 g/L or increasing it to 80 g/L was not effective. In all three genotypes, the absence of temperature pretreatment of buds (5–6 °C) showed the best results. The plant regeneration with MS nutrient medium of 20 g/L sucrose, 3 g/L phytagel, 1 mg/L 6-benzylaminopurine (BAP) and 0.1 mg/L gibberellic acid (GA3) resulted in up to seven shoots from one induced ovule in the most responsive genotype. We showed by flow cytometry, chromosome counting and chloroplast number assessment that all regenerant plants were haploid (2n = x = 9).

Respons Planlet Stevia (Stevia rebaudiana) terhadap Penambahan berbagai Konsentrasi Thidiazuron (TDZ) dan Naphthalene Acetic Acid (NAA) secara in Vitro

Stevia can be developed by vegetative and generative propagation, while the generative propagation of stevia can be done through seeds. Conventional provision of seeds or seedlings has a low success rate. Propagation via tissue culture is faster, produces more seeds, and is identical to the parent, so the plants’ quality is expected. Plant growth regulator (PGR) often used for tissue culture is type auxins, cytokinins and gibberellins. The function of auxin and cytokinin for division cells, cell elongation, cell differentiation and organ formation. This research aims to get the concentration as well as the best combination for the growth of stevia plants against the administration of thidiazuron (TDZ) and naphthalene acetic acid (NAA). Research carried out by the randomised block design (RBD) factorial pattern consisting of two factors: TDZ and NAA. The first factor is TDZ concentration consisting of three levels: 0 mg.l-1 (T0), 1 mg.l-1 (T1), and 2 mg. l-1 (T2). The second factor is NAA concentration, which consists of three levels: 0 mg.l-1 (N0), 1 mg.l-1 (N1), and 2 mg.l-1 (N1). If there is a difference in data analysis using the F test, then proceed with the BNT level test of 5%. Based on the research results obtained, the influence of TDZ growth regulators and no effect of the concentration of NAA growth regulator on stevia bud multiplication book number. There is an effect of TDZ on plantlet height, leaves, and stevia shoot multiplication nodes.

Establishment of a Highly Efficient In Vitro Regeneration System for Rhododendron aureum

Rhododendron aureum is identified as a vulnerable species in China. The establishment of an in vitro regeneration system assists significantly in species protection. Here, an in vitro regeneration system was developed using both direct and indirect organogenesis pathways. The role of thidiazuron (TDZ) in different developmental stages was also investigated. The leaves of wild-harvested R. aureum plants were used for callus induction after hydroponic cultivation. The optimal formula was found to be woody plant basal medium (WPM) supplemented with 0.5 mg·L−1 TDZ and 0.5 mg·L−1 3-indolebutyric acid (IBA), while the optimal formula for the subculture and induction of adventitious buds was WPM containing 0.1 mg·L−1 TDZ and 0.5 mg·L−1 IBA. The leaves from tissue-cultured seedlings were then used for the induction of bud clusters. An association was observed between the differentiation of bud clusters and the ratio of auxin and cytokinin. The optimal formula for the induction of bud clusters was WPM containing 0.5 mg·L−1 TDZ and 0.1 mg·L−1 IBA, yielding a 50% induction rate and the maximum number of buds. Higher concentrations of TDZ were found to be beneficial for bud proliferation, while a lower concentration was conducive to stem elongation. The optimal formula for subculture was WPM containing 0.1 mg·L−1 TDZ and 0.5 mg·L−1 IBA, while that for stem elongation was WPM supplemented with 0.002 mg·L−1 TDZ and 0.5 mg·L−1 IBA. Only IBA was found to be necessary for rooting, with increased IBA concentrations leading to an increased number of roots and earlier root formation, with larger callus areas; thus, an IBA concentration of 1.0 mg·L−1 was found to be optimal for the rooting of R. aureum. After hardening the seedlings for two days, a substrate composed of vermiculite and peat soil in a 10:1 ratio was identified as a transplantation formula. This system provides directions both for the protection of endangered species and the promotion of industrial development.

Thidiazuron Promoted Microspore Embryogenesis and Plant Regeneration in Curly Kale (Brassica oleracea L. convar. acephala var. sabellica)

Curly kale (Brassica oleracea L. convar. acephala var. sabellica), the most common type of edible kale, characterized by providing rich nutrition and health care functions, is sought after and has been listed as top of the healthiest vegetables in recent trends, and has aroused the interest of breeders in cultivating new varieties. However, it usually takes more than six years to obtain a homozygous kale inbred line for commercial seed production through conventional breeding procedures due to its long growth and development period. The isolated microspore culture (IMC) technique could be a time-saving alternative method for producing doubled haploid (DH) lines that are genetically homozygous. In this study, we successfully utilize the efficient cytokinin thidiazuron (TDZ) to promote microspore embryogenesis and plant regeneration in two curly kale cultivars (‘Winterbor F2’ and ‘Starbor F2’). Compared with the control (0 mg/L TDZ), all tested TDZ concentrations (0.1, 0.2, 0.3, 0.4 mg/L) had no adverse effects on embryogenesis, and 0.2 mg/L TDZ had an optimal effect on embryo survival and plant regeneration of the two genotypes. For ‘Starbor F2’, 0.2 mg/L TDZ treatment achieved the highest embryogenesis rate (1.83-fold higher than the control group) and direct seeding rate (1.61-fold increase), and the lowest mortality rate. Likewise, 0.2 mg/L TDZ increased the embryogenesis rate of ‘Winterbor F2’ by 1.62 times, the direct seeding rate by 1.61 times, and the mortality rate fell to the lowest. A 1/2 Murashige and Skoog (MS) medium with 0.2 mg/L 1-Naphthaleneacetic acid (NAA) can significantly promote the rooting of the regenerated seedlings. These results provide new insights into the practical application of the IMC technique in shortening the breeding cycle of kale.

Flowering Time and Physiological Reaction of Dendrobium nobile Lindl in Response to TDZ Application

The objective of this work was to analyze the effect of Thidiazuron (TDZ) treatment on floral initiation, flowering time, ornamental characteristics and physiological metabolism of potted Dendrobium nobile. Three TDZ concentrations (200, 500 and 1000 mg L−1) were applied as solution to water the root zone of the plants. Control plants (plants watered with water) showed a good vegetative development but no floral branches. TDZ greatly influenced the flowering process. For all the tested TDZ concentrations, the first flower bud occurred at 55–60 days after the last irrigation (DAI), the highest TDZ concentration showing the major delay in its occurrence. The initial flowering (30% of flowered plants) began 47 days after the first flower bud initiation with no statistical differences among the treatments. Plants treated with TDZ 500–1000 mg L−1 showed the longest period of flowering (about 32 days) and the single flowers delayed the withering of about 2–3 days compared to the lowest TDZ treatment (200 mg L−1). The number of flowers, floral branches and flowering percentage were distinctly influenced by the TDZ concentration. The highest percentage of flowering (40%) was scored when plants were watered with a TDZ solution at 500 mg L−1 and this was a performant treatment providing the best morphological flower features for the ornamental value of this plant. Among the physiological factors affecting the flowering, this study showed that TDZ increased the relative membrane permeability which facilitated the transport of macromolecular flower-forming substances into and out of the membrane. Therefore, the membrane permeability change could be an indicator of shifts in physiologically active substances during the flowering transition process in Dendrobium nobile plants.

Potential and Constraints on In Vitro Micropropagation of Juniperus drupacea Labill.

Juniperus drupacea Labill. (Cupressaceae) is a species with ecological and medicinal value. In Europe, it is native only in southern Greece, and is listed as endangered. Due to its uniqueness, this study attempted, for the first time, an in vitro propagation effort of Syrian juniper. Explants of the lateral shoot tips were surface-sterilized and cultured on Murashige and Skoog (MS) medium. The cultures were subcultured on MS, woody plant medium (WPM), and Driver and Kuniyaki Walnut (DKW) supplemented with different concentrations of 6-benzylaminopurine (BA), thidiazuron (TDZ), or meta-topolin [6-(3-hy-droxybenzylamino)purine] for shoot induction. Explants derived from female trees exhibited 54.17% bud proliferation on DKW medium with 4 μM meta-topolin or 4 μM TDZ and on WPM with 4 μM meta-topolin or 4 μM BA. A total of 62.50% of the male tree derived explants produced multiple shoots on DKW with 4 μM BA. The maximum average number of shoots per explant were 1.17 per explant in both cases. The length of the shoot derived from explants of female origin was 2.94 mm compared to 2.69 mm of the in vitro shoots from the explants of male trees. Overall, the best medium and plant growth regulator combination for the explants derived from both female and male trees, for the traits under study, was proven to be DKW + 4 µM TDZ. Our experiments show that Juniperus drupacea, under in vitro conditions, shows recalcitrance in rooting, as the applications of IBA, NAA, and IAA concentrations were proven to be ineffective treatments. Although the results show low values, this avant-garde study provides a foundation for further research on the in vitro regeneration of Juniperus drupacea.

Effects of TDZ and IBA on DNA methylation during callus induction in pear (<i>Pyrus ussuriensis</i> Maxim)

One of the essential epigenetic processes in higher eukaryotes, DNA methylation is essential for maintaining genome integrity and influencing gene expression. There aren't many research on how plant growth regulators (PGR) affect DNA methylation during the pear callus formation process. In this work, By using methylation-sensitive amplification polymorphism (MSAP), DNA methylation in pear callus caused by various quantities of thidiazuron (TDZ) and indole-3-butyric acid (IBA) was compared. The concentrations of TDZ and IBA could affect the callus induction rate and methylation level. It was discovered that induction rate and methylation were negatively correlated. The rate of pear callus induction was highest (54%) while the medium suppled with 3.0 mg·L^-1 TDZ and 4.0 mg·L^-1 IBA, although the matching DNA methylation level was lowest (27.96%). Additionally, there were significant difference in th level of genes' expression between different treatments and Both methylation and demethylation are regulated by these genes. As for genes related to auxin and cytokinin, gene expression analysis revealed that their levels of expression after different concentrations of TDZ combined with IBA present in differences significantly. Our findings provide light on DNA methylation mechanisms of plant tissue culture (PTC) dedifferentiation.

Indução de florescimento in vitro de Dendrobium nobile

Abstract In vitro flowering is a technique used in genetic improvement that accelerates generations and favors the faster fixation of new traits of agronomic and market interest. The study aimed to establish a protocol for the in vitro flowering of Dendrobium nobile, through the combined temperature and of the growth regulator thidiazuron (TDZ) effects. Experiments was performed in a flask that was kept in a BOD incubator at 18, 21, or 24 °C or in a growth room at 26 °C. The TDZ concentrations were 0.0, 0.5, 1.0, and 2.0 mg L-1. The highest percentage of flowering shoots of 13.7% occurred at a concentration of 2 mg L-1 of TDZ grown at a temperature of 18 °C. Dendrobium nobile in vitro flowering was promoted in plants cultivated in ½ MS supplemented with 30 g L-1 sucrose, 5.5 g L-1 agar, 100 mL L-1 coconut water, pH 6.0, and 2.0 mg L-1 TDZ at a controlled temperature of 18 °C and a photoperiod of 16 h. The in vitro flowering induction protocol of the D. nobile species could be used or improved for future studies.

Biotechnology of the Tree Fern Cyathea smithii Hook. f. (Soft Tree Fern, Katote). I. Morphogenic Potential of Shoot Apical Dome, Plant Regeneration, and Nuclear DNA Content of Regenerants in the Presence of TDZ and NAA

Abstract In nature, the tree fern Cyathea smithii lacks meristem multiplication; therefore, a system of vegetative propagation in vitro is required to protect the species’ gene resources. Experiments were carried out on the apical dome of C. smithii J. D. Hooker (soft tree fern, Katote) to determine its morphogenic potential using 1/2 strength of Murashige and Skoog medium containing various concentration of thidiazuron [TDZ; 1-phenyl-3-(1,2,3-thiadiazol-5-yl)-urea)] (0.01, 0.1, 1.0, and 5.0 µM) supplemented with naphthalene acetic acid (0.25 µM). The success of the treatments was determined by the number of regenerants per explant, level of regenerant development, and nuclear DNA content in the green mesophyll cells. The highest number of regenerants was achieved in the presence of 5.0 µM TDZ and the lowest at 0.01 µM TDZ. The quality of development of the regenerants was expressed as the number of formed leaves and their growth per individual plantlet. The highest growth and development of regenerants occurred at the lowest concentration of TDZ (0.01 µM), and the lowest growth and development of regenerants occurred at the highest TDZ concentration (5.0 µM); however, the number of regenerants showed the opposite. In gametophytes, the nuclear DNA content ranged from 4.74–4.78 pg representing haploid level, whereas in sporophytes it ranged from 8.45–9.13 pg and was diploid. These results prove the stability of the regenerant genome after TDZ treatment of meristematic cells.

Establishment of Direct Organogenesis Protocol for Arachis hypogaea cv. Virginia in Liquid Medium by Temporary Immersion System (TIS)

Peanuts (Arachis hypogaea L.) are a rich source of herbal oil, proteins, minerals, vitamins, fibers, essential amino acids, as well as bioactive compounds, and are thus widely used for human nutrition and animal feed, and for prevention from certain diseases. However, the in vitro regeneration response of the species is generally low, and it also displays a significant variability among its varieties. Thus, the development of advanced protocols and approaches for the in vitro propagation of peanut is still of immense importance. A recently developed in vitro propagation technique, TIS; Temporary Immersion Bioreactor System, provides a new approach for the mass propagation of plants. Accordingly, the present study provides an efficient de novo regeneration protocol for Arachis hypogaea L. cv. Virginia by using a TIS. Different concentrations of cytokinins, i.e., benzyladenine (BA) or thidiazuron (TDZ), were tested with several combinations of dry and medium immersion periods of TIS, corresponding to a total of 8, 12, 16, 24, 32, 36, 48, 64, 72, and 96 min daily immersions for the induction of direct organogenesis. The study exhibited that an MS medium added to 110 µM BA or 10 µM TDZ are the most appropriate medium formulations in TIS, when applied for 16 min every 16 h. The application of optimized procedures to cv. NC7 and two valuable Turkish autochthonous varieties, 7 × 77 and Com74, is also reported. To the best of our knowledge, the present study draws attention also for being the first study in which a TIS was used for peanuts.

High-frequency Shoot Multiplication and In Vitro Plantlet Regeneration from Shoot Bud Explants in Curcuma zedoaria Roscoe

ABSTRACT A rapid and efficient micropropagation protocol was developed for Curcuma zedoaria, using shoot buds as explants. Explants established on basal MS medium were transferred to 12 different medium compositions with different combinations and concentrations of plant growth hormones 6-benzylaminopurine (BAP), kinetin (Kn), and thidiazuron (TDZ). The best shoot multiplication was recorded in MS medium supplemented with BAP (2.0 mg L−1) and TDZ (1.5 mg L−1), resulting in 5.3 ± 0.24 shoots of per explant and shoot length of 6.12 ± 0.19 cm. For the best in vitro root induction, the aseptic shoots excised from the culture flasks and inoculated individually on MS medium supplemented with 1.5 mg L−1 indole-3-butyric acid (IBA) resulted in 74.2% root growth and 8.82 ± 0.31 roots per explant. Although callus induction was observed in MS containing 2,4-dichlorophenoxyacetic acid (2, 4-D) and 1-naphthaleneacetic acid (NAA), the highest callus proliferation was observed with MS and NAA (2.0 mg L−1). Micropropagated plants established on soil showed 83% survival on acclimatization to the field conditions.

In vitro asymbiotic germination, seedling formation and tuberization of Orchis sancta

Plant tissue culture techniques have recently become attractive tools for propagating rare and endangered species and producing their economically important natural products. Here, we introduce a protocol for an effective, fast and reliable production route of plantlets and tubers from salep orchid, Orchis sancta, a native orchid species growing wild in Turkey. In the course of this study, the efficacy of basal media and plant growth regulators on asymbiotic germination of mature seed, seedling formation, tuberization and glucomannan contents of tubers were assessed. Amongst all basal media evaluated, Orchimax including activated charcoal gave the best germination rate (62.46%). Supplementing this medium with 1.0 mg/L zeatin resulted in the highest germination rate (81.18%). Lowest thidiazuron concentration (0.25 mg/L) was very effective on shoot elongation while kinetin (0.5 and 1.0 mg/L) promoted leaf formation. The aforesaid basal medium with IBA favored root extension (31.16 mm) and root formation (4.5 roots per plantlet) at 1.0 mg/L and 2.0 mg/L IBA, respectively. Cytokinins, zeatin (2.0 mg/L) in particular, promoted tuber formation and glucomannan accumulation. Plantlets were successfully adapted to soil step by step.

Genetic Evaluation of In Vitro Micropropagated and Regenerated Plants of Cannabis sativa L. Using SSR Molecular Markers.

Simple sequence repeat (SSR) markers were used to evaluate the genetic stability of the acclimatized micropropagated and regenerated plants of a high cannabidiol (H-CBD) and a high cannabigerol (H-CBG) variety of Cannabis sativa L. Shoot regeneration and proliferation were achieved by culturing calli in Murashige and Skoog basal medium (MS) supplemented with several concentrations of 6-benzyladenine (BA) or thidiazuron (TDZ). Calli derived mostly from stem explants, rather than leaves, cultured on MS supplemented with 2,4-Dichlorophenoxyacetic acid (2,4-D) or combination of kinetin (KIN) with 1-Naphthaleneacetic acid (NAA) or 2,4-D. Rooting of the regenerated plantlets accomplished on half-strength MS medium supplemented with indole-3-butyric acid (IBA). Previous studies performed have developed an efficient in vitro micropropagation protocol for mass production. Both in vitro methodologies can be employed in genetic breeding via molecular techniques. The genetic stability of micropropagated and regenerated plants was accomplished using twelve SSR primer pairs that produced reproducible and clear bands, ranging from 90 to 330 bp in size, and resulted in amplification of one or two alleles, corresponding to homozygous or heterozygous individuals. The SSR amplification products were monomorphic across all the micropropagated and regenerated plants and comparable to mother plants. The monomorphic banding pattern confirmed the genetic homogeneity of the in vitro cultured acclimatized and mother plants as no somaclonal variation was detected in clones for these specific SSRs. Our results evidently suggest that the developed culture protocols for in vitro multiplication is appropriate and applicable for clonal mass propagation of the C. sativa varieties and demonstrate the reliability of this in vitro propagation system.