Research

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16 pages, 1614 KiB

Open AccessArticle

Phytochemical Composition and Biological Activities of Extracts from Early, Mature, and Germinated Somatic Embryos of Cotyledon orbiculata L.

by Gokhan Zengin, Zoltán Cziáky, József Jekő, Kyung Won Kang, José Manuel Lorenzo and Iyyakkannu Sivanesan

Plants 2023, 12(5), 1065; https://doi.org/10.3390/plants12051065 - 27 Feb 2023

Cited by 1 | Viewed by 1230

Abstract

Cotyledon orbiculata L. (Crassulaceae)—round-leafed navelwort—is used worldwide as a potted ornamental plant, and it is also used in South African traditional medicine. The current work aims to assess the influence of plant growth regulators (PGR) on somatic embryogenesis (SE) in C. orbiculata; [...] Read more.

Cotyledon orbiculata L. (Crassulaceae)—round-leafed navelwort—is used worldwide as a potted ornamental plant, and it is also used in South African traditional medicine. The current work aims to assess the influence of plant growth regulators (PGR) on somatic embryogenesis (SE) in C. orbiculata; compare the metabolite profile in early, mature, and germinated somatic embryos (SoEs) by utilizing ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS); and determine the antioxidant and enzyme inhibitory potentials of SoEs. A maximum SoE induction rate of 97.2% and a mean number of SoEs per C. orbiculata leaf explant of 35.8 were achieved on Murashige and Skoog (MS) medium with 25 µM 2,4-Dichlorophenoxyacetic acid and 2.2 µM 1-phenyl-3-(1,2,3,-thiadiazol-5-yl)urea. The globular SoEs were found to mature and germinate best on MS medium with gibberellic acid (4 µM). The germinated SoE extract had the highest amounts of both total phenolics (32.90 mg gallic acid equivalent/g extract) and flavonoids (1.45 mg rutin equivalent/g extract). Phytochemical evaluation of SoE extracts by UHPLC-MS/MS reveals the presence of three new compounds in mature and germinated SoEs. Among the SoE extracts tested, germinated SoE extract exhibited the most potent antioxidant activity, followed by early and mature somatic embryos. The mature SoE extract showed the best acetylcholinesterase inhibitory activity. The SE protocol established for C. orbiculata can be used for the production of biologically active compounds, mass multiplication, and conservation of this important species. Full article

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

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17 pages, 1173 KiB

Open AccessArticle

Optimizing Medium Composition and Environmental Culture Condition Enhances Antioxidant Enzymes, Recovers Gypsophila paniculata L. Hyperhydric Shoots and Improves Rooting In Vitro

by Suzan M. Mohamed, Mohammed E. El-Mahrouk, Antar N. El-Banna, Yaser M. Hafez, Hassan El-Ramady, Neama Abdalla and Judit Dobránszki

Plants 2023, 12(2), 306; https://doi.org/10.3390/plants12020306 - 09 Jan 2023

Cited by 2 | Viewed by 1927

Abstract

Gypsophila paniculata L. is one of the most important commercial cut flowers worldwide. The plant is sterile and propagated mainly by in vitro culture techniques. However, hyperhydricity hinders its micropropagation and increases mortality during ex vitro acclimatization. Hyperhydric shoots of G. paniculata were [...] Read more.

Gypsophila paniculata L. is one of the most important commercial cut flowers worldwide. The plant is sterile and propagated mainly by in vitro culture techniques. However, hyperhydricity hinders its micropropagation and increases mortality during ex vitro acclimatization. Hyperhydric shoots of G. paniculata were proliferated from nodal explants on MS medium without growth regulators that contained 30 g L⁻¹ sucrose, and gelled with 6.0 g L⁻¹ agar. Medium components and environmental culture conditions were optimized to revert hyperhydricity in G. paniculata microshoots and develop an efficient micropropagation protocol for commercial production. Multiple shoots with high quality were successfully regenerated on MS medium fortified with potassium and ammonium nitrate at full concentration, 2.0 mg L⁻¹ paclobutrazol, solidified with 9.0 g L⁻¹agar in Magenta boxes of 62.87 gas exchange/day and incubated under light density of 60 µmol m⁻²s⁻¹. We recorded 4.33 shoots, 40.00 leaves, 6.33 cm, 2.50 g and 95.00% for number of shoots/explant, number of leaves/shoot, shoot length, shoot fresh weight and normal shoots percentage, respectively. Well-rooted plantlets of G. paniculata were developed from the reverted microshoots, with the rooting percentage (95.00%) on MS medium augmented with 1.0 mg L⁻¹ IBA in Magenta boxes of 62.87 gas exchange/day and 60 µmol m⁻²s⁻¹ light density. In vitro-rooted plantlets exhibited reduced electrolyte leakage, and enhanced antioxidant enzymes activity of peroxidase, catalase, and polyphenol oxidase due to good ventilation at the highest gas exchange rate of the culture vessels. Full article

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

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10 pages, 2095 KiB

Open AccessArticle

Cannabis Synthetic Seeds: An Alternative Approach for Commercial Scale of Clonal Propagation and Germplasm Conservation

by Adel Zarei, Biruk A. Feyissa, Benjamin Davis and Elham Tavakouli Dinani

Plants 2022, 11(23), 3186; https://doi.org/10.3390/plants11233186 - 22 Nov 2022

Cited by 2 | Viewed by 3922

Abstract

Indoor cannabis (Cannabis sativa) cultivation has been rapidly increasing in many countries after legalization. Besides conventional propagation through cuttings, synthetic seed production provides a competent system for mass propagation, germplasm conservation and international exchange of genetic materials. The present study developed [...] Read more.

Indoor cannabis (Cannabis sativa) cultivation has been rapidly increasing in many countries after legalization. Besides conventional propagation through cuttings, synthetic seed production provides a competent system for mass propagation, germplasm conservation and international exchange of genetic materials. The present study developed a reliable protocol for cannabis synthetic seed production using encapsulation of nodal segments derived from in vitro or in vivo sources. Synthetic seeds were produced in 3% sodium alginate and 75 mM calcium chloride in Murashige and Skoog (MS) medium and stored under various environmental conditions for up to 150 days. The plantlets regrowth efficiency was monitored on culture media up to 30 days after the storage period. Regrowth rates of 70% and 90% were observed in synthetic seeds from in vitro and in vivo-derived sources, respectively, when stored in 6 °C under 50 μmol s⁻¹ m⁻² light for 150 days. Furthermore, addition of acetylsalicylic acid (ASA) to the encapsulation matrix not only postponed precocious germination of synthetic seeds at 22 °C, but also improved the regrowth rate of in vivo-derived synthetic seeds to 100% when they were stored in 6 °C under light. Exposure to light during storage significantly increased shoot length of regrown synseeds when compared to those stored in darkness. This difference in shoot growth disappeared when synseeds were treated with 25 µM ASA. All regenerated plantlets were rooted and acclimatized in sterile rockwool plugs without morphological changes. Full article

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

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14 pages, 2542 KiB

Open AccessArticle

Establishing an Efficient Regeneration System for Tissue Culture in Bougainvillea buttiana ‘Miss Manila’

by Tao Huang, Huihui Zhang, Runan Zhao and Zunling Zhu

Plants 2022, 11(18), 2372; https://doi.org/10.3390/plants11182372 - 11 Sep 2022

Cited by 8 | Viewed by 2615

Abstract

Bougainvillea plants have important ornamental and cultural value, as well as applications, for example, in improving the ecological environment, sterilization and as anti-virals in medicines, etc. Among many varieties, Bougainvillea buttiana ‘Miss Manila’ is more popular in landscape applications in southern China because [...] Read more.

Bougainvillea plants have important ornamental and cultural value, as well as applications, for example, in improving the ecological environment, sterilization and as anti-virals in medicines, etc. Among many varieties, Bougainvillea buttiana ‘Miss Manila’ is more popular in landscape applications in southern China because of its excellent qualities. However, because of the difficulty of sexual reproduction, low rooting rate of asexual propagation cuttings and low temperature tolerance, its large-scale development is limited. For easy, quick and mass multiplication of such cultivars, tissue culture technique can be put to use. In this study, nodal segments of ‘Miss Manila’ were used as explants, and a single-factor experiment was carried out with a variety of plant growth regulators and concentrations to establish and optimize a complete tissue culture propagation system. The results showed that the best disinfestation was 75% ethanol treatment for 0.5 min + 0.1% HgCl₂ treatment for 9 min, and the survival rate was 86.83%. The best shoot initiation formula was MS + 6-BA (2.5 mg/L) + IBA (0.2 mg/L), and the induction rate was 95.14%. The best formula for shoot proliferation was MS + 6-BA (1.5 mg/L) + NAA (0.1 mg/L), and the proliferation coefficient was 3.13. The best rooting culture formula was 1/2 MS + IBA (2.0 mg/L), and the rooting rate was 81.97%. The survival rate for plantlet refining and transplanting was 93.33%. In this study, a relatively efficient regeneration system for the tissue culture and rapid propagation of Bougainvillea buttiana ‘Miss Manila’ was established to address the problems of rooting and transplanting of this variety, to benefit research on the industrialized production and regeneration systems of this variety, and to provide a reference for the tissue culture of other varieties of Bougainvillea or other woody plants. Full article

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

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19 pages, 2108 KiB

Open AccessArticle

Callus Induction from Diverse Explants and Genotypes Enables Robust Transformation of Perennial Ryegrass (Lolium perenne L.)

by Daniel Grogg, Marius Rohner, Steven Yates, Chloe Manzanares, Simon E. Bull, Sue Dalton, Maurice Bosch, Bruno Studer and Giovanni A. L. Broggini

Plants 2022, 11(15), 2054; https://doi.org/10.3390/plants11152054 - 05 Aug 2022

Cited by 4 | Viewed by 3380

Abstract

Genetic transformation of perennial ryegrass (Lolium perenne L.) is critical for fundamental and translational research in this important grass species. It often relies on Agrobacterium-mediated transformation of callus tissue. However, callus induction is restricted to a few genotypes that respond well [...] Read more.

Genetic transformation of perennial ryegrass (Lolium perenne L.) is critical for fundamental and translational research in this important grass species. It often relies on Agrobacterium-mediated transformation of callus tissue. However, callus induction is restricted to a few genotypes that respond well to tissue culture. Here, we report callus induction from different perennial ryegrass genotypes and explants, such as shoot tips, seeds, and anthers, which were transformed with several plasmids for functional genomics. β-glucuronidase (GUS) histochemical staining showed the LmdsRNAbp promoter sequence was active in stigmas, spikelets, anthers, and leaves. We also transformed calli with plasmids allowing gene silencing and gene knock-out using RNA interference and CRISPR/Cas9, respectively, for which genotypic and phenotypic investigations are ongoing. Using 19 different constructs, 262 transgenic events were regenerated. Moreover, the protocol regenerated a doubled haploid transgenic event from anther-derived calli. This work provides a proof-of-concept method for expanding the range of genotypes amenable to transformation, thus, serving research and breeding initiatives to improve this important grass crop for forage and recreation. Full article

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

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18 pages, 3430 KiB

Open AccessArticle

Enhanced Carbonylation of Photosynthetic and Glycolytic Proteins in Antibiotic Timentin-Treated Tobacco In Vitro Shoot Culture

by Elena Andriūnaitė, Rytis Rugienius, Inga Tamošiūnė, Perttu Haimi, Jurgita Vinskienė and Danas Baniulis

Plants 2022, 11(12), 1572; https://doi.org/10.3390/plants11121572 - 14 Jun 2022

Cited by 1 | Viewed by 1587

Abstract

Antibiotics are used in plant in vitro tissue culture to eliminate microbial contamination or for selection in genetic transformation. Antibiotic timentin has a relatively low cytotoxic effect on plant tissue culture; however, it could induce an enduring growth-inhibiting effect in tobacco in vitro [...] Read more.

Antibiotics are used in plant in vitro tissue culture to eliminate microbial contamination or for selection in genetic transformation. Antibiotic timentin has a relatively low cytotoxic effect on plant tissue culture; however, it could induce an enduring growth-inhibiting effect in tobacco in vitro shoot culture that persists after tissue transfer to a medium without antibiotic. The effect is associated with an increase in oxidative stress injury in plant tissues. In this study, we assessed changes of reactive oxygen species accumulation, protein expression, and oxidative protein modification response associated with enduring timentin treatment-induced growth suppression in tobacco (Nicotiana tabacum L.) in vitro shoot culture. The study revealed a gradual 1.7 and 1.9-fold increase in superoxide (O₂^•−) content at the later phase of the propagation cycle for treatment control (TC) and post-antibiotic treatment (PA) shoots; however, the O₂^•− accumulation pattern was different. For PA shoots, the increase in O₂^•− concentration occurred several days earlier, resulting in 1.2 to 1.4-fold higher O₂^•− concentration compared to TC during the period following the first week of cultivation. Although no protein expression differences were detectable between the TC and PA shoots by two-dimensional electrophoresis, the increase in O₂^•− concentration in PA shoots was associated with a 1.5-fold increase in protein carbonyl modification content after one week of cultivation, and protein carbonylation analysis revealed differential modification of 26 proteoforms involved in the biological processes of photosynthesis and glycolysis. The results imply that the timentin treatment-induced oxidative stress might be implicated in nontranslational cellular redox balance regulation, accelerates the development of senescence of the shoot culture, and contributes to the shoot growth-suppressing effect of antibiotic treatment. Full article

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

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11 pages, 2346 KiB

Open AccessArticle

Adventitious Shoot Regeneration from Leaf Explants in Sinningia Hybrida ‘Isa’s Murmur’

by Honglin Yang, Yihua Yang, Qiang Wang, Jinyu He, Liyun Liang, Hui Qiu, Yue Wang and Lijuan Zou

Plants 2022, 11(9), 1232; https://doi.org/10.3390/plants11091232 - 02 May 2022

Cited by 5 | Viewed by 2540

Abstract

As a valuable ornamental plant, Sinningia hybrida ‘Isa’s Murmur’ (S. hybrida) has genetic flower diversity, which has great potential to develop different flower characters in the horticultural market. The present study focuses on establishing a practical approach for the sustainable propagation [...] Read more.

As a valuable ornamental plant, Sinningia hybrida ‘Isa’s Murmur’ (S. hybrida) has genetic flower diversity, which has great potential to develop different flower characters in the horticultural market. The present study focuses on establishing a practical approach for the sustainable propagation of S. hybrida. Compared with aseptic seeding leaves explants, field-grown leaves explants are more suitable for adventitious shoot regeneration. Adding 0.1 mg L⁻¹ NAA and 2.0 mg L⁻¹ TDZ could obtain the highest adventitious shoot proliferation coefficient (24.5), and the induction rate was 91.7%. The shoot proliferation coefficient (20.7) and the greatest shoot length and induction rate (95.3%) were achieved in 0.1 mg L⁻¹ NAA and 2.0 mg L⁻¹ BA medium, accompanied by rooting formation. Adding 0.5 mg L⁻¹ GA₃, 1.0 mg L⁻¹ BA, and 0.2 mg L⁻¹ IBA to MS medium can effectively prolong the regenerated buds for rooting. The best for rooting was 1/2 MS medium containing 0.3 mg L⁻¹ IBA, with the maximum number of roots (13.4 per shoot) and survival rate for transplanting (100%). This work aims to build an efficient, definitive, and scalable protocol for S. hybrida regeneration useful for large-scale cultivation and even more protoplast fusion and genetic transformation to develop more colorful or fragrant flowers. Full article

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

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10 pages, 2453 KiB

Open AccessCommunication

In Vitro Propagation of Humulus lupulus through the Induction of Axillary Bud Development

by Takeshi Hirakawa and Seia Tanno

Plants 2022, 11(8), 1066; https://doi.org/10.3390/plants11081066 - 13 Apr 2022

Cited by 5 | Viewed by 2532

Abstract

Humulus lupulus (hop) is a necessary material for beer brewing. Improved breeding cultivars of hops with enhanced tolerance to environmental stresses, such as drought and heat stress, accompanying climate change have been developed. However, a propagation system, which is needed for the proliferation [...] Read more.

Humulus lupulus (hop) is a necessary material for beer brewing. Improved breeding cultivars of hops with enhanced tolerance to environmental stresses, such as drought and heat stress, accompanying climate change have been developed. However, a propagation system, which is needed for the proliferation of new cultivars, is not currently available for hops. In this study, we found that treatment of stem explants with 0.01–0.05 ppm gibberellic acid (GA₃) induced the development of axillary buds in the hop cultivar Kirin-2, resulting in the proliferation of shoot branching. Additionally, 0.01 ppm benzyl adenine (BA) enhanced the development of axillary buds formed in response to 0.05 ppm GA₃ in various hop cultivars, particularly Nugget. The development of axillary buds was strongly repressed by the application of 0.05 ppm BA at a concentration equal to the 0.05 ppm GA₃ concentration, which showed the possibility that a high concentration of cytokinin preferentially prevents the effect of GA₃ on the development of axillary buds in hops. These results indicated that combined treatment of stem explants with GA₃ and cytokinin at appropriate concentrations is effective for the propagation of proliferated hop cultivars through shoot branching. Full article

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

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18 pages, 12680 KiB

Open AccessArticle

Enduring Effect of Antibiotic Timentin Treatment on Tobacco In Vitro Shoot Growth and Microbiome Diversity

by Inga Tamošiūnė, Elena Andriūnaitė, Jurgita Vinskienė, Vidmantas Stanys, Rytis Rugienius and Danas Baniulis

Plants 2022, 11(6), 832; https://doi.org/10.3390/plants11060832 - 21 Mar 2022

Cited by 3 | Viewed by 2677

Abstract

Plant in vitro cultures initiated from surface-sterilized explants often harbor complex microbial communities. Antibiotics are commonly used to decontaminate plant tissue culture or during genetic transformation; however, the effect of antibiotic treatment on the diversity of indigenous microbial populations and the consequences on [...] Read more.

Plant in vitro cultures initiated from surface-sterilized explants often harbor complex microbial communities. Antibiotics are commonly used to decontaminate plant tissue culture or during genetic transformation; however, the effect of antibiotic treatment on the diversity of indigenous microbial populations and the consequences on the performance of tissue culture is not completely understood. Therefore, the aim of this study was to assess the effect of antibiotic treatment on the growth and stress level of tobacco (Nicotiana tabacum L.) shoots in vitro as well as the composition of the plant-associated microbiome. The study revealed that shoot cultivation on a medium supplemented with 250 mg L⁻¹ timentin resulted in 29 ± 4% reduced biomass accumulation and a 1.2–1.6-fold higher level of oxidative stress injury compared to the control samples. Moreover, the growth properties of shoots were only partially restored after transfer to a medium without the antibiotic. Microbiome analysis of the shoot samples using multivariable region-based 16S rRNA gene sequencing revealed a diverse microbial community in the control tobacco shoots, including 59 bacterial families; however, it was largely dominated by Mycobacteriaceae. Antibiotic treatment resulted in a decline in microbial diversity (the number of families was reduced 4.5-fold) and increased domination by the Mycobacteriaceae family. These results imply that the diversity of the plant-associated microbiome might represent a significant factor contributing to the efficient propagation of in vitro tissue culture. Full article

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

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Review

Jump to: Research

12 pages, 320 KiB

Open AccessReview

Hyperhydricity in Plant Tissue Culture

by Oksana B. Polivanova and Vladislav A. Bedarev

Plants 2022, 11(23), 3313; https://doi.org/10.3390/plants11233313 - 30 Nov 2022

Cited by 17 | Viewed by 3958

Abstract

Hyperhydricity is the most common physiological disorder in in vitro plant cultivation. It is characterized by certain anatomical, morphological, physiological, and metabolic disturbances. Hyperhydricity significantly complicates the use of cell and tissue culture in research, reduces the efficiency of clonal micropropagation and the [...] Read more.

Hyperhydricity is the most common physiological disorder in in vitro plant cultivation. It is characterized by certain anatomical, morphological, physiological, and metabolic disturbances. Hyperhydricity significantly complicates the use of cell and tissue culture in research, reduces the efficiency of clonal micropropagation and the quality of seedlings, prevents the adaptation of plants in vivo, and can lead to significant losses of plant material. This review considers the main symptoms and causes of hyperhydricity, such as oxidative stress, impaired nitrogen metabolism, and the imbalance of endogenous hormones. The main factors influencing the level of hyperhydricity of plants in vitro are the mineral and hormonal composition of a medium and cultivation conditions, in particular the aeration of cultivation vessels. Based on these factors, various approaches are proposed to eliminate hyperhydricity, such as varying the mineral and hormonal composition of the medium, the use of exogenous additives, aeration systems, and specific lighting. However, not all methods used are universal in eliminating the symptoms of hyperhydricity. Therefore, the study of hyperhydricity requires a comprehensive approach, and measures aimed at its elimination should be complex and species-specific. Full article

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

21 pages, 345 KiB

Open AccessReview

Incorporation of Organic Growth Additives to Enhance In Vitro Tissue Culture for Producing Genetically Stable Plants

by Imtinene Hamdeni, Mounir Louhaichi, Slim Slim, Abdennacer Boulila and Taoufik Bettaieb

Plants 2022, 11(22), 3087; https://doi.org/10.3390/plants11223087 - 14 Nov 2022

Cited by 5 | Viewed by 2598

Abstract

The growing demand for native planting material in ecological restoration and rehabilitation for agro-silvo-pastoral ecosystems has resulted in a major global industry in their sourcing, multiplication, and sale. Plant tissue culture is used for producing high-quality, disease-free, and true-to-type plants at a fast [...] Read more.

The growing demand for native planting material in ecological restoration and rehabilitation for agro-silvo-pastoral ecosystems has resulted in a major global industry in their sourcing, multiplication, and sale. Plant tissue culture is used for producing high-quality, disease-free, and true-to-type plants at a fast rate. Micropropagation can help to meet the increasing demand for planting material and afforestation programs. However, in vitro plant propagation is an expensive technique compared to conventional methods using suckers, seeds, and cuttings. Therefore, adopting measures to lower production costs without compromising plant quality is essential. This can be achieved by improving the culture media composition. Incorporating organic growth additives can stimulate tissue growth and increase the number of shoots, leaves, and roots in culture media. Organic growth supplementation speeds up the formation and development of cultures and yields vigorous plants. Plant regeneration from meristems (shoot tips and axillary buds) is a reliable way to produce true-to-type plants compared with callus and somatic embryogenesis regeneration, but in vitro culture environments can be mutagenic. Therefore, detecting somaclonal variations at an early stage of development is considered crucial in propagating plants. The genetic stability of in vitro regenerated plants needs to be ascertained by using DNA-based molecular markers. This review aims to provide up-to-date research progress on incorporating organic growth additives to enhance in vitro tissue culture protocols and to emphasize the importance of using PCR-based molecular markers such as RAPD, ISSR, SSR, and SCoT. The review was assessed based on the peer-reviewed works published in scientific databases including Science Direct, Scopus, Springer, JSTOR, onlinelibrary, and Google Scholar. Full article

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

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