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10 pages, 2454 KiB

Open AccessArticle

Flavonoid Accumulation in an Aseptic Culture of Summer Savory (Satureja hortensis L.)

by Darya A. Khlebnikova, Evgeniya M. Efanova, Nina A. Danilova, Yaroslava V. Shcherbakova and Irina Rivera Sidorova

Plants 2022, 11(4), 533; https://doi.org/10.3390/plants11040533 - 16 Feb 2022

Cited by 3 | Viewed by 2626

Abstract

Summer savory (Satureja hortensis L.) is a medicinal and aromatic plant of the Lamiaceae family, a source of valuable secondary metabolites (monoterpenoids, rosmarinic acid, flavonoids). For this paper, flavonoid accumulation in an aseptic culture of summer savory was determined by using a [...] Read more.

Summer savory (Satureja hortensis L.) is a medicinal and aromatic plant of the Lamiaceae family, a source of valuable secondary metabolites (monoterpenoids, rosmarinic acid, flavonoids). For this paper, flavonoid accumulation in an aseptic culture of summer savory was determined by using a colorimetric method. The organ specificity of flavonoid accumulation in aseptic plants was revealed: In leaves (8.35 ± 0.17 mg/g FW), flower buds (7.55 ± 0.29 mg/g FW), and calyx (5.27 ± 0.28 mg/g FW), flavonoids accumulated in significantly higher amounts than in stems (1.50 ± 0.22 mg/g FW) and corolla (0.78 ± 0.12 mg/g FW). We found that primary callus tissue formed from cotyledon and hypocotyl explants retains the ability to synthesize flavonoids at deficient levels (0.50 ± 0.09 mg/g FW and 0.44 ± 0.11 mg/g FW, respectively), that remained stable throughout six subcultures. Placing the callus tissue in monochrome lighting conditions with blue, green, and red light-emitting diode (LED) lamps leads to morphological changes in the tissue and decreased flavonoid accumulation compared to fluorescent lamps. Full article

(This article belongs to the Special Issue Plant Tissue Culture II)

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12 pages, 2697 KiB

Open AccessArticle

A Temporary Immersion System Improves Regeneration of In Vitro Irradiated Recalcitrant Indica Rice (Oryza sativa L.) Embryogenic Calli

by Alejandro Hernández-Soto, Jason Pérez, Rebeca Fait-Zúñiga, Randall Rojas-Vásquez, Andrés Gatica-Arias, Walter Vargas-Segura and Ana Abdelnour-Esquivel

Plants 2022, 11(3), 375; https://doi.org/10.3390/plants11030375 - 29 Jan 2022

Cited by 5 | Viewed by 4036

Abstract

The development of gamma ray-mutated rice lines is a solution for introducing genetic variability in indica rice varieties already being used by farmers. In vitro gamma ray (⁶⁰Co) mutagenesis reduces chimeras and allows for a faster selection of desirable traits but [...] Read more.

The development of gamma ray-mutated rice lines is a solution for introducing genetic variability in indica rice varieties already being used by farmers. In vitro gamma ray (⁶⁰Co) mutagenesis reduces chimeras and allows for a faster selection of desirable traits but requires the optimization of the laboratory procedure. The objectives of the present work were sequencing of matK and rbcL, the in vitro establishment of recalcitrant rice embryogenic calli, the determination of their sensitivity to gamma radiation, and optimization of the generation procedure. All sequenced genes matched perfectly with previously reported matK and rbcL O. sativa genes. Embryogenic calli induction improved using MS medium containing 2 mg L⁻¹ 2,4-D, and regeneration was achieved with MS medium with 3 mg L⁻¹ BA and 0.5 mg L⁻¹ NAA. The optimized radiation condition was 60 Gy, (LD20 = 64 Gy) with 83% regeneration. An immersion system (RITA^®, Saint-Mathieu-de-Tréviers, France) of either 60 or 120 s every 8 h allowed systematic and homogeneous total regeneration of the recalcitrant line. Other well-known recalcitrant cultivars, CR1821 and CR1113, also had improved regeneration in the immersion system. To our knowledge, this is the first study reporting the use of an immersion system to allow for the regeneration of gamma-ray mutants from recalcitrant indica rice materials. Full article

(This article belongs to the Special Issue Plant Tissue Culture II)

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13 pages, 3384 KiB

Open AccessArticle

Expression Analysis of Phenylpropanoid Pathway Genes and Metabolomic Analysis of Phenylpropanoid Compounds in Adventitious, Hairy, and Seedling Roots of Tartary Buckwheat

by Minsol Choi, Ramaraj Sathasivam, Bao Van Nguyen, Nam Il Park, Sun-Hee Woo and Sang Un Park

Plants 2022, 11(1), 90; https://doi.org/10.3390/plants11010090 - 28 Dec 2021

Cited by 12 | Viewed by 3193

Abstract

Tartary buckwheat (Fagopyrum tataricum) is an important crop that belongs to the Polygonaceae family, whose roots have received considerable attention due to the presence of compounds with high nutritional and medicinal value. In this study, we aimed to develop an efficient [...] Read more.

Tartary buckwheat (Fagopyrum tataricum) is an important crop that belongs to the Polygonaceae family, whose roots have received considerable attention due to the presence of compounds with high nutritional and medicinal value. In this study, we aimed to develop an efficient protocol for the culture of adventitious (ARs) and hairy (HRs) roots on a half-strength Schenk and Hildebrandt (SH) medium containing different concentrations of the auxins, α-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA), and indole-3-acetic acid (IAA). The highest percentage of root induction (91.67%) was achieved with 0.5 mg/L IAA, whereas the greatest number of roots was found in 1 mg/L IAA. In contrast, 0.1 mg/L IBA returned the longest roots. As expected, HRs were obtained from in vitro leaf explants infected with Agrobacterium rhizogenes R1000. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of 11 phenolic pathway genes revealed that five genes (FtPAL, FtC3H, FtHQT, FtCHS, and FtANS) were highly expressed in HRs, whereas only four (FtC4H, FtFLS2, FtDFR, and FtANR), and three (Ft4CL, FtCHI, and FtF3H) were recognized in the ARs and seedling roots (SRs), respectively. HPLC analysis of phenolic compounds in different root cultures showed that the majority of the phenolic compounds (both individual and total) were significantly accumulated in the HRs. Principal component analysis (PCA) identified differences among the three root types, whereby HRs were separated from ARs and SRs based on the amount of phenolic compounds present. Analysis of the metabolic pathway revealed that among the identified metabolites, the 3, 2, and 1 pathways were associated with flavonoid, flavone and flavonol, and phenylpropanoid biosynthesis, respectively. Hierarchical clustering analysis and the heat map showed that the different root cultures presented unique metabolites. Full article

(This article belongs to the Special Issue Plant Tissue Culture II)

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20 pages, 3280 KiB

Open AccessArticle

High-Frequency Plant Regeneration, Genetic Uniformity, and Flow Cytometric Analysis of Regenerants in Rutachalepensis L.

by Ahmed A. Qahtan, Mohamad Faisal, Abdulrahman A. Alatar and Eslam M. Abdel-Salam

Plants 2021, 10(12), 2820; https://doi.org/10.3390/plants10122820 - 20 Dec 2021

Cited by 9 | Viewed by 4861

Abstract

Ruta chalepensis L., an evergreen shrub in the citrus family, is well-known around the world for its essential oils and variety of bioactivities, indicating its potential medicinal applications. In this study, we investigated the effect of different culture conditions, including plant growth regulators, [...] Read more.

Ruta chalepensis L., an evergreen shrub in the citrus family, is well-known around the world for its essential oils and variety of bioactivities, indicating its potential medicinal applications. In this study, we investigated the effect of different culture conditions, including plant growth regulators, media types, pH of the medium, and carbon sources, on in vitro regeneration from nodal explants of R. chalepensis. Following 8 weeks of culture, the highest percentage of regeneration (96.3%) and maximum number of shoots (40.3 shoot/explant) with a length of 4.8 cm were obtained with Murashige and Skoog (MS) medium at pH 5.8, supplemented with 3.0% sucrose and 5.0 µM 6-Benzyladenine (BA) in combination with 1.0 µM 1-naphthaleneacetic acid (NAA). For rooting, individually harvested shootlets were transferred on ½ MS (half-strength) supplemented with IAA (indole-3-acetic acid), IBA (indole 3-butyric acid), or NAA, and the best response in terms of root induction (91.6%), number of roots (5.3), and root mean length (4.9 cm) was achieved with 0.5 µM IBA after 6 weeks. An average of 95.2 percent of healthy, in vitro regenerated plantlets survived after being transplanted into potting soil, indicating that they were effectively hardened. DNA assays (PCR-based markers) such as random amplification of polymorphic DNA (RAPD) and directed amplification of minisatellite-region (DAMD) were employed to assess in vitro cultivated R. chalepensis plantlets that produced a monomorphic banding pattern confirming the genetic stability. Additionally, no changes in the flow cytometric profile of ploidy between regenerated plantlets and donor plants were detected. Regeneration of this valuable medicinal plant in vitro will open up new avenues in pharmaceutical biotechnology by providing an unconventional steadfast system for mass multiplication and might be effectively used in genetic manipulation for enhanced bioactive constituents. Full article

(This article belongs to the Special Issue Plant Tissue Culture II)

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11 pages, 1285 KiB

Open AccessArticle

Differing In Vitro Rooting and Flowering Responses of the Persian Violet to Low and High UV-C Irradiation

by Saowaros Phanomchai, Sompoch Noichinda, Yongsak Kachonpadungkitti and Kitti Bodhipadma

Plants 2021, 10(12), 2671; https://doi.org/10.3390/plants10122671 - 4 Dec 2021

Cited by 3 | Viewed by 2670

Abstract

Persian violet flowers are considered esthetically attractive, leading to the high economic value of this plant. Plant breeding is fundamental to crop improvement, and the induction of mutation by tissue culture technology in combination with irradiation has been beneficially applied to generate plants [...] Read more.

Persian violet flowers are considered esthetically attractive, leading to the high economic value of this plant. Plant breeding is fundamental to crop improvement, and the induction of mutation by tissue culture technology in combination with irradiation has been beneficially applied to generate plants with novel desirable characteristics. In this research, single or double rounds of UV-C irradiations were carried out on plant tissue cultures to initiate the in vitro rooting and mutation of Persian violets. It was found that single low-intensity UV-C exposure, when applied to Persian violet microshoots for 4 h, could induce the maximum number of roots and the highest root length without the use of a plant growth regulator. Overall, the single and double UV-C irradiation of Persian violet microshoots led to 44 different types of Persian violet flower mutations. Under single high-intensity UV-C irradiation for 6 h, up to nine petals were initiated, whereas single low-intensity UV-C irradiation did not influence the morphological variation of Persian violet flowers. Thus, Persian violet microshoots respond differently in terms of in vitro rooting and flowering depending on the UV-C intensity and exposure duration. These outcomes may be applied to micropropagation and in vitro plant breeding. Full article

(This article belongs to the Special Issue Plant Tissue Culture II)

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9 pages, 1138 KiB

Open AccessCommunication

Optimization of Protoplast Isolation from Leaf Mesophylls of Chinese Cabbage (Brassica rapa ssp. pekinensis) and Subsequent Transfection with a Binary Vector

by Ganeshan Sivanandhan, Solhee Bae, Chaemin Sung, Su-Ryun Choi, Geung-Joo Lee and Yong-Pyo Lim

Plants 2021, 10(12), 2636; https://doi.org/10.3390/plants10122636 - 30 Nov 2021

Cited by 15 | Viewed by 3321

Abstract

Chinese cabbage is an important dietary source of numerous phytochemicals, including glucosinolates and anthocyanins. The selection and development of elite Chinese cabbage cultivars with favorable traits is hindered by a long breeding cycle, a complex genome structure, and the lack of an efficient [...] Read more.

Chinese cabbage is an important dietary source of numerous phytochemicals, including glucosinolates and anthocyanins. The selection and development of elite Chinese cabbage cultivars with favorable traits is hindered by a long breeding cycle, a complex genome structure, and the lack of an efficient plant transformation protocol. Thus, a protoplast transfection-based transformation method may be useful for cell-based breeding and functional studies involving Chinese cabbage plants. In this study, we established an effective method for isolating Chinese cabbage protoplasts, which were then transfected with the pCAMBIA1303 binary vector according to an optimized PEG-based method. More specifically, protoplasts were isolated following a 4 h incubation in a solution comprising 1.5% (v/v) cellulase, 0.25% (v/v) macerozyme, 0.25% (v/v) pectinase, 0.5 M mannitol, 15 mM CaCl₂, 25 mM KCl, 0.1% BSA, and 20 mM MES buffer, pH 5.7. This method generated 7.1 × 10⁶ protoplasts, 78% of which were viable. The gfp reporter gene in pCAMBIA1303 was used to determine the transfection efficiency. The Chinese cabbage protoplast transfection rate was highest (68%) when protoplasts were transfected with the 40

μ

g binary vector for 30 min in a solution containing 40% PEG. The presence of gusA and hptII in the protoplasts was confirmed by PCR. The methods developed in this study would be useful for DNA-free genome editing as well as functional and molecular investigations of Chinese cabbage. Full article

(This article belongs to the Special Issue Plant Tissue Culture II)

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17 pages, 4457 KiB

Open AccessArticle

Hypolignification: A Decisive Factor in the Development of Hyperhydricity

by Nurashikin Kemat, Richard G. F. Visser and Frans A. Krens

Plants 2021, 10(12), 2625; https://doi.org/10.3390/plants10122625 - 29 Nov 2021

Cited by 11 | Viewed by 2468

Abstract

One of the characteristics of hyperhydric plants is the reduction of cell wall lignification (hypolignification), but how this is related to the observed abnormalities of hyperhydricity (HH), is still unclear. Lignin is hydrophobic, and we speculate that a reduction in lignin levels leads [...] Read more.

One of the characteristics of hyperhydric plants is the reduction of cell wall lignification (hypolignification), but how this is related to the observed abnormalities of hyperhydricity (HH), is still unclear. Lignin is hydrophobic, and we speculate that a reduction in lignin levels leads to more capillary action of the cell wall and consequently to more water in the apoplast. p-coumaric acid is the hydroxyl derivative of cinnamic acid and a precursor for lignin and flavonoids in higher plant. In the present study, we examined the role of lignin in the development of HH in Arabidopsis thaliana by checking the wild-types (Ler and Col-0) and mutants affected in phenylpropanoid biosynthesis, in the gene coding for cinnamate 4-hydroxylase, C4H (ref3-1 and ref3-3). Exogenously applied p-coumaric acid decreased the symptoms of HH in both wild-type and less-lignin mutants. Moreover, the results revealed that exogenously applied p-coumaric acid inhibited root growth and increased the total lignin content in both wild-type and less-lignin mutants. These effects appeared to diminish the symptoms of HH and suggest an important role for lignin in HH. Full article

(This article belongs to the Special Issue Plant Tissue Culture II)

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12 pages, 2342 KiB

Open AccessArticle

Soybean Callus—A Potential Source of Tocopherols

by Liliana Mureșan, Doina Clapa, Teodor Rusu, Thomas T. Y. Wang and Jae B. Park

Plants 2021, 10(12), 2571; https://doi.org/10.3390/plants10122571 - 25 Nov 2021

Cited by 3 | Viewed by 2815

Abstract

In vitro cultures have been used as an effective means to achieve a high level of secondary metabolites in various plants, including soy. In this study, the contents of α-, γ-, and δ- tocopherol were quantified in soybean callus, and their amounts were [...] Read more.

In vitro cultures have been used as an effective means to achieve a high level of secondary metabolites in various plants, including soy. In this study, the contents of α-, γ-, and δ- tocopherol were quantified in soybean callus, and their amounts were compared to those of soybeans cultivated using the conventional tillage system with three weed controls (respectively without herbicide and with two variants of herbicide). Soybean callus was produced using Murashige and Skoog 1962 (MS) medium supplemented with 0.1 mg/L 6-Benzylaminopurine (BAP) and 0. 1 mg/L Thidiazuron (TDZ). The highest amount of fresh callus was obtained from soybeans from the conventional tillage system with second weed control (S-metolachlor 960 g/L, imazamox 40 g/L, and propaquizafop 100 g/L) respectively 13,652.4 ± 1177.62 mg. The analyzed tocopherols were in much higher content in soy dry callus than the soybean seeds (5.63 µg/g compared with the 0.35 α-toco in soybean, 47.57 µg/g compared with 18.71 µg/g γ-toco or, 5.56 µg/g compared with 1.74 µg/g β-toco). The highest content of the three analyzed tocopherols was γ -tocopherol, both in callus and soybeans. Furthermore, the data showed that herbicides used in soybean culture significantly influenced both the in vitro callus production and the tocopherol callus content (p ˂ 0.05). Altogether, soybean callus can be an important source of tocopherols, and herbicides significantly influence in vitro callus production and the tocopherol callus content. Full article

(This article belongs to the Special Issue Plant Tissue Culture II)

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13 pages, 2229 KiB

Open AccessArticle

A Novel and Efficient In Vitro Organogenesis Approach for Ajuga lupulina Maxim

by Qinggui Wu, Honglin Yang, Yulin Yang, Jinyu He, Erga Aer, Yonghong Ma and Lijuan Zou

Plants 2021, 10(9), 1918; https://doi.org/10.3390/plants10091918 - 15 Sep 2021

Cited by 3 | Viewed by 2282

Abstract

This work was aimed at establishing an effective approach for in vitro propagation of Ajuga lupulina Maxim, a medicinal and ornamental plant mainly found in eastern Xizang, in the western Sichuan region of China. We report an optimum response in the proliferation of [...] Read more.

This work was aimed at establishing an effective approach for in vitro propagation of Ajuga lupulina Maxim, a medicinal and ornamental plant mainly found in eastern Xizang, in the western Sichuan region of China. We report an optimum response in the proliferation of axillary shoots from nodal segment explants (10.2 shoots/explant) on MS medium containing 3.0 mg L⁻¹ of 6-benzyladenine (BA). When BA and TDZ individually or in combination with NAA were employed for adventitious shoot regeneration, shoots and embryo-like structures (ELSs) were noted in the callus from leaf explants. The maximum response of 26.4 shoots /explant (81.6%) and 12.0 ELSs/explant were ascertained on MS medium with 4.0 mg L⁻¹ TDZ and 0.1 mg L⁻¹ NAA. The leaf despite browning still demonstrated a high regeneration capacity. TDZ (2.0 mg L⁻¹) and BA (2.0 mg L⁻¹) along with NAA (0.01 mg L⁻¹) were found to perform well for shoot regeneration via callus from shoot tip explants. The best for rooting was MS medium (half-strength) containing indole-3-butyric acid (IBA: 1.5 mg L⁻¹) and (NAA: 0.5 mg L⁻¹) with the maximum number of roots (25.8 per shoot) and the highest rooting frequency (81.71%). The survival of the plantlets in the greenhouse was 78.2% indicative of successful acclimatization. This work is the first report of a consistent, definitive, and unique protocol for A. lupulina regeneration, paving the way for the in vitro preservation of such significant genetic resources and also further allied systems based on such callus-based or embryo-based approaches. Full article

(This article belongs to the Special Issue Plant Tissue Culture II)

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16 pages, 2671 KiB

Open AccessArticle

In Vitro Propagation and Acclimatization of Banana Plants: Antioxidant Enzymes, Chemical Assessments and Genetic Stability of Regenerates as a Response to Copper Sulphate

by Doaa M. Abou Elyazid, Abdel-Moety Salama, Abdel Fattah M. El Zanaty and Neama Abdalla

Plants 2021, 10(9), 1853; https://doi.org/10.3390/plants10091853 - 7 Sep 2021

Cited by 8 | Viewed by 5480

Abstract

Developing a successful protocol for banana in vitro culture is a guarantee for the mass propagation of pathogen-free, high-quality, true-to-type planting materials with low production costs. The current work aimed to investigate the influence of increasing copper levels in an MS medium on [...] Read more.

Developing a successful protocol for banana in vitro culture is a guarantee for the mass propagation of pathogen-free, high-quality, true-to-type planting materials with low production costs. The current work aimed to investigate the influence of increasing copper levels in an MS medium on endophytic bacterial contamination; shoot multiplication; rooting and the acclimatization of in vitro cultured banana; minerals and chlorophyll content; antioxidant enzymes activity; electrolyte leakage; and the genetic stability of banana regenerants. Four different concentrations of copper sulphate (0.025 as a control, and 30, 60, and 120 mg L⁻¹) were examined. The growth of the endophytic bacteria was inhibited at 60 mg L⁻¹ of copper sulphate which recorded zero contamination, without a significant difference at 120 mg L⁻¹. However, 0.025 mg L⁻¹ of copper sulphate was optimal for the maximum shoot number and shoot length (10 shoots and 6 cm, respectively) without significant differences at 30 mg L⁻¹. The root length of banana plantlets was significantly enhanced at 30 mg L⁻¹ of copper sulphate but without significant differences to the control, regarding the number of roots (9.92 cm and 3.80 roots, respectively). In vitro plants were acclimatized successfully at 30 mg L⁻¹ of copper sulphate with 100% survival. The uptake of minerals, antioxidant enzyme activity and electrolyte leakage was improved because of the copper sulphate, but the chlorophyll level decreased. RAPD profiling showed polymorphism in only one plant treated with 60 mg L⁻¹ of copper sulphate, with an average of 1.8%. The genome template stability percentage was almost 100% for all treated plants. Full article

(This article belongs to the Special Issue Plant Tissue Culture II)

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16 pages, 2914 KiB

Open AccessArticle

In Vitro Production and Exudation of 20-Hydroxymaytenin from Gymnosporia heterophylla (Eckl. and Zeyh.) Loes. Cell Culture

by Thanet Pitakbut, Michael Spiteller and Oliver Kayser

Plants 2021, 10(8), 1493; https://doi.org/10.3390/plants10081493 - 21 Jul 2021

Cited by 1 | Viewed by 2825

Abstract

The metabolite 20-Hydroxymaytenin (20-HM) is a member of the quinone-methide pentacyclic triterpenoids (QMTs) group. This metabolite group is present only in Celastraceae plants, and it has shown various biological activities from antioxidant to anticancer properties. However, most QMTs metabolites including 20-HM cannot be [...] Read more.

The metabolite 20-Hydroxymaytenin (20-HM) is a member of the quinone-methide pentacyclic triterpenoids (QMTs) group. This metabolite group is present only in Celastraceae plants, and it has shown various biological activities from antioxidant to anticancer properties. However, most QMTs metabolites including 20-HM cannot be synthesized in a laboratory. Therefore, we optimized a plant tissue culture protocol and examined the potential of Gymnosporia heterophylla (synonym. Maytenus heterophylla) to produce 20-HM in an in vitro experiment. For the first time, we reported the optimum callus induction medium with a high percentage success rate of 82% from the combination of 1 mg/L indole-3-butyric acid and 5 mg/L naphthalene acetic acid. Later, our cell suspension culture cultivated in the optimum medium provided approximately 0.35 mg/g fresh weight of 20-HM. This concentration is roughly 87.5 times higher than a concentration of 20-HM presenting in Elaeodendron croceum (Celastraceae) leaves. In addition, we also found that 20-HM presented in a cultivation medium, suggesting that G. heterophylla cells secreted 20-HM as an exudate in our experiment. Noticeably, 20-HM was missing when Penicillium cf. olsonii occurred in the medium. These findings hint at an antifungal property of 20-HM. Full article

(This article belongs to the Special Issue Plant Tissue Culture II)

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10 pages, 1843 KiB

Open AccessCommunication

Protocols for Adventitious Regeneration of Amelanchier alnifolia var. cusickii and Lonicera kamtschatica ‘Jugana’

by Júlia Hunková, Monika Szabóová and Alena Gajdošová

Plants 2021, 10(6), 1155; https://doi.org/10.3390/plants10061155 - 6 Jun 2021

Cited by 5 | Viewed by 2847

Abstract

The aim of this work was to assess the regeneration capacity of Amelanchier alnifolia var. cusickii and Lonicera kamtschatica cv. ‘Jugana’ from different types of explants under various hormonal treatments. The whole leaves, petioles, and internodal segments of in vitro plants were examined [...] Read more.

The aim of this work was to assess the regeneration capacity of Amelanchier alnifolia var. cusickii and Lonicera kamtschatica cv. ‘Jugana’ from different types of explants under various hormonal treatments. The whole leaves, petioles, and internodal segments of in vitro plants were examined as explants. Several plant growth regulators (cytokinins and auxins) were evaluated for their ability to induce adventitious regeneration. Direct and indirect organogenesis was achieved under certain culture conditions in both species. The frequency of shoot regeneration was strongly dependent on concentrations of plant growth regulators in the induction media (L.kamtschatica ‘Jugana’) or concentrations of plant growth regulators in the induction media and type of explant (A. alnifolia var. cusickii). Results showed that leaves were not suitable explants for A. alnifolia var. cusickii. Both species were able to regenerate shoots from internodal segments and petioles. The highest induction of shoots was obtained on Murashige and Skoog (MS) medium enriched with 2 mg/L thidiazuron (TDZ) and 0.5 mg/L indole-3-butyric acid (IBA) for Amelanchier alnifolia and with 1 mg/L TDZ and 0.2 mg/L indole-3-acetic acid (IAA) for L. kamtschatica ‘Jugana’. Obtained adventitious shoots were further proliferated in order to investigate their multiplication capacity. The multiplication of shoots was successful in all cultivars, with the best results reported in A. alnifolia var. cusickii (7.07 shoots/explant on average). Full article

(This article belongs to the Special Issue Plant Tissue Culture II)

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19 pages, 5641 KiB

Open AccessArticle

A More Accessible, Time-Saving, and Efficient Method for In Vitro Plant Regeneration from Potato Protoplasts

by Ki-Beom Moon, Ji-Sun Park, Su-Jin Park, Hyo-Jun Lee, Hye-Sun Cho, Sung-Ran Min, Youn-Il Park, Jae-Heung Jeon and Hyun-Soon Kim

Plants 2021, 10(4), 781; https://doi.org/10.3390/plants10040781 - 16 Apr 2021

Cited by 11 | Viewed by 5991

Abstract

Both obtaining high-yielding, viable protoplasts and following reliable regeneration protocols are prerequisites for the continuous expansion and development of newly emerging systems involving protoplast utilization. This study determines an efficient process from protoplast isolation to shoot regeneration in vitro. The maximum yield of [...] Read more.

Both obtaining high-yielding, viable protoplasts and following reliable regeneration protocols are prerequisites for the continuous expansion and development of newly emerging systems involving protoplast utilization. This study determines an efficient process from protoplast isolation to shoot regeneration in vitro. The maximum yield of protoplast extraction, which was 6.36 ± 0.51 × 10⁶ protoplasts/g fresh weight (FW), was approximately 3.7 times higher than that previously reported for potato protoplasts. To obtain data, wounded leaves were used by partially cutting both sides of the midrib, and isolated protoplasts were purified by the sucrose cushion method, with a sucrose concentration of 20%. We confirmed a significant effect on the extraction efficiency by measuring enzymolysis during a 6 h period, with three times more washing buffer than the amount normally used. Protoplasts fixed in alginate lenses with appropriate space were successfully recovered and developed into microcalli 2 weeks after culture. In addition, to induce high efficiency regeneration from protoplasts, calli in which greening occurred for 6 weeks were induced to develop shoots in regeneration medium solidified by Gelrite, and they presented a high regeneration efficiency of 86.24 ± 11.76%. Full article

(This article belongs to the Special Issue Plant Tissue Culture II)

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24 pages, 3305 KiB

Open AccessArticle

Establishment of a Rapid Micropropagation System for Kaempferia parviflora Wall. Ex Baker: Phytochemical Analysis of Leaf Extracts and Evaluation of Biological Activities

by Han-Yong Park, Kyung-Su Kim, Gunes Ak, Gokhan Zengin, Zoltán Cziáky, József Jekő, Kathalingam Adaikalam, Kihwan Song, Doo-Hwan Kim and Iyyakkannu Sivanesan

Plants 2021, 10(4), 698; https://doi.org/10.3390/plants10040698 - 5 Apr 2021

Cited by 23 | Viewed by 5072

Abstract

This study aimed to establish a rapid in vitro plant regeneration method from rhizome buds of Kaempferia parviflora to obtain the valuable secondary metabolites with antioxidant and enzyme inhibition properties. The disinfection effect of silver oxide nanoparticles (AgO NPs) on rhizome and effects [...] Read more.

This study aimed to establish a rapid in vitro plant regeneration method from rhizome buds of Kaempferia parviflora to obtain the valuable secondary metabolites with antioxidant and enzyme inhibition properties. The disinfection effect of silver oxide nanoparticles (AgO NPs) on rhizome and effects of plant growth regulators on shoot multiplication and subsequent rooting were investigated. Surface sterilization of rhizome buds with sodium hypochlorite was insufficient to control contamination. However, immersing rhizome buds in 100 mg L⁻¹ AgO NPs for 60 min eliminated contamination without affecting the survival of explants. The number of shoots (12.2) produced per rhizome bud was higher in Murashige and Skoog (MS) medium containing 8 µM of 6-Benzyladenine (6-BA) and 0.5 µM of Thidiazuron (TDZ) than other treatments. The highest number of roots (24), with a mean root length of 7.8 cm and the maximum shoot length (9.8 cm), were obtained on medium MS with 2 µM of Indole-3-butyric acid (IBA). A survival rate of 98% was attained when plantlets of K. parviflora were acclimatized in a growth room. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) was used to determine the chemical profile of K. parviflora leaf extracts. Results showed that several biologically active flavonoids reported in rhizomes were also present in leaf tissues of both in vitro cultured and ex vitro (greenhouse-grown) plantlets of K. parviflora. We found 40 and 36 compounds in in vitro cultured and ex vitro grown leaf samples, respectively. Greenhouse leaves exhibited more potent antioxidant activities than leaves from in vitro cultures. A higher acetylcholinesterase inhibitory ability was obtained for greenhouse leaves (1.07 mg/mL). However, leaves from in vitro cultures exhibited stronger butyrylcholinesterase inhibitory abilities. These results suggest that leaves of K. parviflora, as major byproducts of black ginger cultivation, could be used as valuable alternative sources for extracting bioactive compounds. Full article

(This article belongs to the Special Issue Plant Tissue Culture II)

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12 pages, 5254 KiB

Open AccessArticle

Development of a Cell Suspension Culture System for Promoting Alkaloid and Vinca Alkaloid Biosynthesis Using Endophytic Fungi Isolated from Local Catharanthus roseus

by Tran My Linh, Nguyen Chi Mai, Pham Thi Hoe, Ninh Thi Ngoc, Phan Thi Hong Thao, Ninh Khac Ban and Nguyen Tuong Van

Plants 2021, 10(4), 672; https://doi.org/10.3390/plants10040672 - 31 Mar 2021

Cited by 22 | Viewed by 4155

Abstract

Cell and tissue cultures of Catharanthus roseus have been studied extensively as an alternative strategy to improve the production of valuable secondary metabolites. The purpose of this study was to produce C. roseus callus and suspension cell biomass of good quality and quantity [...] Read more.

Cell and tissue cultures of Catharanthus roseus have been studied extensively as an alternative strategy to improve the production of valuable secondary metabolites. The purpose of this study was to produce C. roseus callus and suspension cell biomass of good quality and quantity to improve the total alkaloids and bis-indole alkaloids. The young stem derived-callus of C. roseus variety Quang Ninh (QN) was grown on MS medium supplemented with 1.5 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) plus 1.5 mg/L kinetin, and the growth rate increased by 67-fold after 20 days. The optimal conditions for maintaining the cell suspension culture were 150 mg/50 mL cell inoculum, a medium pH of 5.5 and a culture temperature of 25 °C. The low alkaloid content in the culture was compensated for by using endophytic fungi isolated from local C. roseus. Cell extracts of endophytic fungi—identified as Fusarium solani RN1 and Chaetomium funicola RN3—were found to significantly promote alkaloid accumulation. This elicitation also stimulated the accumulation of a tested bis-indole alkaloid, vinblastine. The findings are important for investigating the effects of fungal elicitors on the biosynthesis of vinblastine and vincristine, as well as other terpenoid indole alkaloids (TIAs), in C. roseus QN cell suspension cultures. Full article

(This article belongs to the Special Issue Plant Tissue Culture II)

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17 pages, 968 KiB

Open AccessArticle

Micropropagation of Ginger (Zingiber officinale Roscoe) ‘Bentong’ and Evaluation of Its Secondary Metabolites and Antioxidant Activities Compared with the Conventionally Propagated Plant

by Nisar Ahmad Zahid, Hawa Z. E. Jaafar and Mansor Hakiman

Plants 2021, 10(4), 630; https://doi.org/10.3390/plants10040630 - 26 Mar 2021

Cited by 36 | Viewed by 13035

Abstract

‘Bentong’ ginger is the most popular variety of Zingiber officinale in Malaysia. It is vegetatively propagated and requires a high proportion of rhizomes as starting planting materials. Besides, ginger vegetative propagation using its rhizomes is accompanied by several types of soil-borne diseases. Plant [...] Read more.

‘Bentong’ ginger is the most popular variety of Zingiber officinale in Malaysia. It is vegetatively propagated and requires a high proportion of rhizomes as starting planting materials. Besides, ginger vegetative propagation using its rhizomes is accompanied by several types of soil-borne diseases. Plant tissue culture techniques have been applied in many plant species to produce their disease-free planting materials. As ‘Bentong’ ginger is less known for its micropropagation, this study was conducted to investigate the effects of Clorox (5.25% sodium hypochlorite (NaOCl)) on explant surface sterilization, effects of plant growth regulators, and basal media on shoots’ multiplication and rooting. The secondary metabolites and antioxidant activities of the micropropagated plants were evaluated in comparison with conventionally propagated plants. Rhizome sprouted buds were effectively sterilized in 70% Clorox for 30 min by obtaining 75% contamination-free explants. Murashige and Skoog (MS) supplemented with 10 µM of zeatin was the suitable medium for shoot multiplication, which resulted in the highest number of shoots per explant (4.28). MS medium supplemented with 7.5 µM 1-naphthaleneacetic acid (NAA) resulted in the highest number of roots per plantlet. The in vitro-rooted plantlets were successfully acclimatized with a 95% survival rate in the ex vitro conditions. The phytochemical analysis showed that total phenolic acid and total flavonoid content and antioxidant activities of the micropropagated plants were not significantly different from the conventionally propagated plants of ‘Bentong’ ginger. In conclusion, the present study’s outcome can be adopted for large-scale propagation of disease-free planting materials of ‘Bentong’ ginger. Full article

(This article belongs to the Special Issue Plant Tissue Culture II)

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10 pages, 1851 KiB

Open AccessArticle

Metabolic Engineering of Isoflavonoid Biosynthesis by Expressing Glycine max Isoflavone Synthase in Allium cepa L. for Genistein Production

by Ashwini Malla, Balamurugan Shanmugaraj, Balamurugan Srinivasan, Ashutosh Sharma and Sathishkumar Ramalingam

Plants 2021, 10(1), 52; https://doi.org/10.3390/plants10010052 - 29 Dec 2020

Cited by 12 | Viewed by 3124

Abstract

Isoflavonoids, the diverse group of secondary metabolites derived from the phenylpropanoid pathway, are distributed predominantly in leguminous plants and play a vital role in promoting human health. Genetic engineering of the metabolite synthesis pathway has turned out to be an attractive approach for [...] Read more.

Isoflavonoids, the diverse group of secondary metabolites derived from the phenylpropanoid pathway, are distributed predominantly in leguminous plants and play a vital role in promoting human health. Genetic engineering of the metabolite synthesis pathway has turned out to be an attractive approach for the production of various secondary metabolites. In our study, we attempted to produce the isoflavone genistein, a well-known health-promoting metabolite, in Allium cepa L. (onion) by introducing Glycine max Isoflavone synthase (GmIFS). The GmIFS gene was cloned into the pEarleyGate 102 HA vector and transformed into onion by Agrobacterium-mediated and biolistic methods. The presence of GmIFS in transgenic onion was confirmed by PCR, dot blot, and Southern hybridization. Analysis of the transgenic onion calli lines demonstrated that the expression of the GmIFS gene led to the production of isoflavone genistein in in vitro tissues. The biolistic stable transformed calli with transformation efficiency of 73% (62.65 nM/g FW) accumulated more genistein than the Agrobacterium stable transformed calli with transformation efficiency of 56% (42.5 nM/g FW). Overall, heterologous gene expression of GmIFS was demonstrated by modifying the secondary metabolite pathway in onion tissues for the production of isoflavone genistein that can boost up human health with its health-promoting properties. Full article

(This article belongs to the Special Issue Plant Tissue Culture II)

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Review

Jump to: Research

27 pages, 34437 KiB

Open AccessReview

Tissue Culture Response of Ornamental and Medicinal Aesculus Species—A Review

by Snežana Zdravković-Korać, Jelena Milojević, Maja Belić and Dušica Ćalić

Plants 2022, 11(3), 277; https://doi.org/10.3390/plants11030277 - 20 Jan 2022

Cited by 4 | Viewed by 3209

Abstract

Species of the genus Aesculus are very attractive woody ornamentals. Their organs contain numerous health-promoting phytochemicals. The most valuable of them—aescin—is used in commercial preparations for the treatment of venous insufficiency. The industrial source of aescin is horse chestnut seeds because the zygotic [...] Read more.

Species of the genus Aesculus are very attractive woody ornamentals. Their organs contain numerous health-promoting phytochemicals. The most valuable of them—aescin—is used in commercial preparations for the treatment of venous insufficiency. The industrial source of aescin is horse chestnut seeds because the zygotic embryos are the main site of its accumulation. Horse chestnut somatic and zygotic embryos contain similar amount of aescin, hence somatic embryos could be exploited as an alternative source of aescin. Somatic embryogenesis, androgenesis and de novo shoot organogenesis were successfully achieved in several Aesculus species, as well as secondary somatic embryogenesis and shoot organogenesis, which enables mass production of embryos and shoots. In addition, an efficient method for cryopreservation of embryogenic tissue was established, assuring constant availability of the plant material. The developed methods are suitable for clonal propagation of elite specimens selected as the best aescin producers, the most attractive ornamentals or plants resistant to pests and diseases. These methods are also useful for molecular breeding purposes. Thus, in this review, the medicinal uses and a comprehensive survey of in vitro propagation methods established for Aesculus species, as well as the feasibility of in vitro production of aescin, are presented and discussed. Full article

(This article belongs to the Special Issue Plant Tissue Culture II)

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15 pages, 1559 KiB

Open AccessReview

Bulb Dormancy In Vitro—Fritillaria meleagris: Initiation, Release and Physiological Parameters

by Marija Marković, Milana Trifunović Momčilov, Branka Uzelac, Slađana Jevremović and Angelina Subotić

Plants 2021, 10(5), 902; https://doi.org/10.3390/plants10050902 - 30 Apr 2021

Cited by 15 | Viewed by 4019

Abstract

In ornamental geophytes, conventional vegetative propagation is not economically feasible due to very slow development and ineffective methods. It can take several years until a new plant is formed and commercial profitability is achieved. Therefore, micropropagation techniques have been developed to increase the [...] Read more.

In ornamental geophytes, conventional vegetative propagation is not economically feasible due to very slow development and ineffective methods. It can take several years until a new plant is formed and commercial profitability is achieved. Therefore, micropropagation techniques have been developed to increase the multiplication rate and thus shorten the multiplication and regeneration period. The majority of these techniques rely on the formation of new bulbs and their sprouting. Dormancy is one of the main limiting factors to speed up multiplication in vitro. Bulbous species have a period of bulb dormancy which enables them to survive unfavorable natural conditions. Bulbs grown in vitro also exhibit dormancy, which has to be overcome in order to allow sprouting of bulbs in the next vegetation period. During the period of dormancy, numerous physiological processes occur, many of which have not been elucidated yet. Understanding the process of dormancy will allow us to speed up and improve breeding of geophytes and thereby achieve economic profitability, which is very important for horticulture. This review focuses on recent findings in the area of bulb dormancy initiation and release in fritillaries, with particular emphasis on the effect of plant growth regulators and low-temperature pretreatment on dormancy release in relation to induction of antioxidative enzymes’ activity in vitro. Full article

(This article belongs to the Special Issue Plant Tissue Culture II)

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